Escherichia coli strains which over-produce L-thereonine and processes for the production of L-threonine by fermentation

ABSTRACT

The present invention relates to the fields of microbiology and microbial genetics. More specifically, the invention relates to novel bacterial strains and processes employing these strains for the fermentative production of amino acids such as threonine.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This non-provisional application is related to provisionalapplication numbered 60/235,884, filed Sep. 28, 2000, the content ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the fields of microbiology andmicrobial genetics. More specifically, the invention relates to novelbacterial strains and processes employing these strains for fermentativeproduction of amino acids such as L-threonine.

[0004] 2. Related Art

[0005] In Escherichia coli, the amino acids L-threonine, L-isoleucine,L-lysine and L-methionine derive all or part of their carbon atoms fromaspartate (aspartic acid) via the following common biosynthetic pathway(G. N. Cohen, “The Common Pathway to Lysine, Methionine and Threonine,”pp. 147-171 in Amino Acids: Biosynthesis and Genetic Regulation, K. M.Herrmann and R. L. Somerville, eds., Addison-Welesley Publishing Co.,Inc., Reading, Mass. (1983)):

[0006] The first reaction of this common pathway is catalyzed by one ofthree distinct aspartate kinases (AK I, II, or III), each of which isencoded by a separate gene and differs from the others in the way itsactivity and synthesis are regulated.

[0007] Aspartate kinase I, for example, is encoded by thrA, its activityis inhibited by threonine, and its synthesis is repressed by threonineand isoleucine in combination. AK II, however, is encoded by metL andits synthesis repressed by methionine (although its activity is notinhibited by methionine or by paired combinations of methionine, lysine,threonine and isoleucine (F. Falcoz-Kelly et al., Eur. J. Biochem.8:146-152 (1969); J. C. Patte et al., Biochim. Biophys. Acta 136:245-257(1967)). AK III is encoded by lysCand its activity and synthesis areinhibited and repressed, respectively, by lysine.

[0008] Two of the AKs, I and II, are not distinct proteins, but rather adomain of a complex enzyme that includes homoserine dehydrogenase I orII, respectively, each of which catalyzes the reduction of aspartatesemialdehyde to homoserine (P. Truffa-Bachi et al., Eur. J. Biochem.5:73-80 (12968)). Homoserine dehydrogenase I (HD I) is therefore alsoencoded by thrA, its synthesis is repressed by threonine plus isoleucineand its activity is inhibited by threonine. Homoserine dehydrogenase II(HD II) is similarly encoded by metL and its synthesis is repressed bymethionine.

[0009] Threonine biosynthesis includes the following additionalreactions:

[0010] Homoserine→Homoserine Phosphate→Threonine. The phosphorylation ofhomoserine is catalyzed by homoserine kinase, a protein which iscomposed of two identical 29 kDa subunits encoded for by thrB and whoseactivity is inhibited by threonine (B. Burr et al., J. Biochem.62:519-526 (1976)). The final step, the complex conversion of homoserinephosphate to L-threonine is catalyzed by threonine synthase, a 47 kDaprotein encoded for by thrc (C. Parsot et al., Nucleic Acids Res.11:7331-7345 (1983)).

[0011] Isoleucine can be produced in E. coli using threonine as aprecursor (see Hashiguchi et al., Biosci. Biotechnol. Biochem.63:672-679 (1999). More specifically, isoleucine is produced via thefollowing reactions:

[0012] Threonine→α-Ketobutyrate→α-Aceto-α-Hydroxybutyrate→α,β-Dihydroxy-β-Methylvalerate α-Keto-β-Methylvalerate→Isoleucine. Thesereactions are catalyzed in E. coli, respectively, by the followingenzymes: threonine deaminase (ilvA); aceto-hydroxyacid synthetase I, II,or III (ilvBN, ilvGM, and ilvIH, respectively); dihydroxyacidreductoisomerase (ilvC); dihydroxyacid dehydratase (ilvD); andtransaminase-B (ilvE).

[0013] The E. coli isoleucine operon is composed of ilvA, ilvGM, ilvD,and ilvE.

[0014] The ilvA gene product (i. e., threonine deaminase) is inhibitedby L-isoleucine, and the ilvGM gene product (i.e., aceto-hydroxyacidsynthetase II) is inhibited by L-valine. Further, the reactionscatalyzed by threonine deaminase and the aceto-hydroxyacid synthetasesare believed to be the main rate limiting steps in the production ofisoleucine.

[0015] The thrA, thrB and thrC genes all belong to the thr operon, asingle operon located at 0 minutes on the genetic map of E. coli (J.Thèze and I. Saint-Girons, J. Bacteriol. 118:990-998 (1974); J. Thèze etal., J. Bacteriol. 11 7:133-143 (1974)). These genes encode,respectively, for aspartate kinase I-homoserine dehydrogenase I,homoserine kinase and threonine synthase. Biosynthesis of these enzymesis subject to multivalent repression by threonine and isoleucine (M.Freundlich, Biochem. Biophys. Res. Commun. 10:277-282 (1963)).

[0016] A regulatory region is found upstream of the first structuralgene in the thr operon and its sequence has been determined (J. F.Gardner, Proc. Natl. Acad. Sci. USA 76:1706-1710 (1979)). The thrattenuator, downstream of the transcription initiation site, contains asequence encoding a leader peptide; this sequence includes eightthreonine codons and four isoleucine codons. The thr attenuator alsocontains the classical mutually exclusive secondary structures whichpermit or prevent RNA polymerase transcription of the structural genesin the thr operon, depending on the levels of the charged threonyl- andisoleucyl-tRNAs.

[0017] Because of the problems associated with obtaining high levels ofamino acid production via natural biosynthesis (e.g., repression of thethr operon by the desired product), bacterial strains have been producedhaving plasmids containing a thr operon with a thrA gene that encodes afeedback-resistant enzyme. With such plasmids, L-threonine has beenproduced on an industrial scale by fermentation processes employing awide variety of microorganisms, such as Brevibacterium flavum, Serratiamarcescens, and E. coli.

[0018] For example, the E. coli strain BKIIM B-3996 (Debabov et al.,U.S. Pat. No. 5,175,107), which contains the plasmid pVIC40, makes about85 g/L in 36 hr. The host is a threonine-requiring strain because of adefective threonine synthase. In BKIIEM B-3996, it is the recombinantplasmid, pVIC40, that provides the crucial enzymatic activities, i.e., afeedback-resistant AK I-HD I, homoserine kinase and threonine synthase,needed for threonine biosynthesis. This plasmid also complements thehost's threonine auxotrophy.

[0019]E. coli strain 29-4 (E. Shimizu et al., Biosci. Biotech. Biochem.59:1095-1098 (1995)) is another example of a recombinant E. colithreonine producer. Strain 29-4 was constructed by cloning the throperon of a threonine-over-producing mutant strain, E. coli K-12 (βIM-4)(derived from E. coli strain ATCC Deposit No. 21277), into plasmidpBR322, which was then introduced into the parent strain (K. Wiwa etal., Agric. Biol. Chem. 47:2329-2334 (1983)). Strain 29-4 produces about65 g/L of L-threonine in 72 hr.

[0020] Similarly constructed recombinant strains have been made usingother organisms. For example, the Serratia marcescens strain T2000contains a plasmid having a thr operon which encodes afeedback-resistant thrA gene product and produces about 100 g/L ofthreonine in 96 hrs (M. Masuda et al., Applied Biochem. Biotechn.37:255-262 (1992)). All of these strains contain plasmids havingmultiple copies of the genes encoding the threonine biosyntheticenzymes, which allows over-expression of these enzymes. Thisover-expression of the plasmid-borne genes encoding threoninebiosynthetic enzymes, particularly a thrA gene encoding afeedback-resistant AK I-HD I, enables these strains to produce largeamounts of threonine. Other examples of plasmid-containingmicroorganisms are described, for example, in U.S. Pat. Nos. 4,321,325;4,347,318; 4,371,615; 4,601,983; 4,757,009; 4,945,058; 4,946,781;4,980,285; 5,153,123; and 5,236,831.

[0021] Plasmid-containing strains such as those described above,however, have problems that limit their usefulness for commercialfermentative production of amino acids. For example, a significantproblem with these strains is ensuring that the integrity of theplasmid-containing strain is maintained throughout the fermentationprocess because of potential loss of the plasmid during cell growth anddivision. To avoid this problem, it is necessary to selectivelyeliminate plasmid-free cells during culturing, such as by employingantibiotic resistance genes on the plasmid. This solution, however,necessitates the addition of one or more antibiotics to the fermentationmedium, which is not commercially practical for large scalefermentations

[0022] Another significant problem with plasmid-containing strains isplasmid stability. High expression of enzymes whose genes are coded onthe plasmid, which is necessary for commercially practical fermentativeprocesses, often brings about plasmid instability (E. Shimizu et al.,Biosci. Biotech. Biochem. 59:1095-1098 (1995)). Plasmid stability isalso dependent upon factors such as cultivation temperature and thelevel of dissolved oxygen in the culture medium.

[0023] For example, plasmid-containing strain 29-4 was more stable atlower cultivation temperatures (30° C. vs. 37° C.) and higher levels ofdissolved oxygen (E. Shimizu et al., Biosci. Biotech. Biochem.59:1095-1098 (1995)).

[0024] Non-plasmid containing microorganisms, while less efficaciousthan those described above, have also been used as threonine producers.Strains of E. coli such as H-8460, which is obtained by a series ofconventional mutagenesis and selection for resistance to severalmetabolic analogs makes about 75 g/L of L-threonine in 70 hours (Kino etal., U.S. Pat. No. 5,474,918). Strain H-8460 does not carry arecombinant plasmid and has one copy of the threonine biosynthetic geneson the chromosome. The lower productivity of this strain compared to theplasmid-bearing strains, such as BKIIM B-3996, is believed to be due tolower enzymatic activities (particularly those encoded by the throperon) as these non-plasmid containing strains carry only a single copyof threonine biosynthetic genes.

[0025] An L-threonine producing strain of E. coli, KY10935, produced bymultiple rounds of mutation is described in K. Okamoto et al., Biosci.Biotechnol. Biochem. 61:1877-1882 (1997). When cultured under optimalconditions with DL-methionine, strain KY10935 is reported to produce asmuch as 100 g/liter L-threonine after 77 hours of cultivation. The highlevel of L-threonine produced is believed to result from the inabilityof this strain to take up L-threonine that accumulates extracellularly,resulting in a decrease in the steady-state level of intracellularL-threonine and the release the remaining regulatory steps in theL-threonine production pathway from feedback inhibition.

[0026] Other examples of suitable non-plasmid containing microorganismsare described, for example, in U.S. Pat. Nos. 5,939,307; 5,474,918;5,264,353; 5,164,307; 5,098,835; 5,087,566; 5,077,207; 5,017,483;4,463,094; 3,580,810; and 3,375,173.

[0027] In both the non-plasmid and plasmid containing strains of E.coli, the thr operon is controlled by the particular strain's respectivenative threonine promoter. As described above, the expression of thenative promoter is regulated by an attenuation mechanism controlled by aregion of DNA which encodes a leader peptide and contains a number ofthreonine and isoleucine codons. This region is translated by a ribosomewhich senses the levels of threoninyl-tRNA and isoleucinyl-tRNA. Whenthese levels are sufficient for the leader peptide to be translated,transcription is prematurely terminated, but when the levels areinsufficient for the leader peptide to be translated, transcription isnot terminated and the entire operon is transcribed, which, followingtranslation, results in increased production of the threoninebiosynthetic enzymes. Thus, when threonyl-tRNA and/or isoleucinyl-tRNAlevels are low, the thr operon is maximally transcribed and thethreonine biosynthetic enzymes are maximally made.

[0028] In the E. coli threonine-producing strain BKIIM B-3996, thethreonine operon in the plasmid is controlled by its native promoter. Asa result, the thr operon is only maximally expressed when the strain isstarved for threonine and/or isoleucine. Since starvation for threonineis not possible in a threonine-producing strain, these strains have beenrendered auxotrophic for isoleucine in order to obtain a higher level ofenzymatic activity.

[0029] Another way of overcoming attenuation control is to lower thelevel(s) of threonyl-tRNA and/or isoleucinyl-tRNA in the cell. A thrSmutant, for example, having a threonyl-tRNA synthase which exhibits a200-fold decreased apparent affinity for threonine, results inover-expression of the thr operon, presumably due to the low level ofthreonyl-tRNA (E. J. Johnson et al., J. Bacteriol., 129:66-70 (1977)).

[0030] In fermentation processes using these strains, however, the cellsmust be supplemented with isoleucine in the growth stage because oftheir deficient isoleucine biosynthesis. Subsequently, in the productionstage, the cells are deprived of isoleucine to induce expression of thethreonine biosynthetic enzymes. A major drawback, therefore, of usingnative threonine promoters to control expression of the threoninebiosynthetic enzymes is that the cells must be supplemented withisoleucine.

[0031] The antibiotic borrelidin, a natural product of Streptomycesrochei, is also known to reduce the enzymatic activity of threonyltRNA-synthetase, and thereby inhibit the growth of E. coli (G. Nass etal., Biochem. Biophys. Res. Commun. 34:84(1969)). In view of thisreduced activity, certain borrelidin-sensitive strains of E. coli havebeen employed to produce high levels of threonine (Japanese PublishedPatent Application No.6752/76; U.S. Pat. No.5,264,353). Addition ofborrelidin to the culture was found to increase the yield ofL-threonine. Borrelidin-sensitive strains of Brevibacterium andCorynebacterium have also been used to produce high levels of threonine(Japanese Patent No. 53- 101591).

[0032] Borrelidin-resistant mutants of E. coli similarly exhibit changesin threonyl tRNA-synthestase activity. More specifically,borrelidin-resistant E. coli have been shown to exhibit one of thefollowing features: (i) constitutively increased levels of wild-typethreonyl tRNA-synthetase; (ii) structurally altered threonyltRNA-synthetase; or (iii) some unknown cellular alteration, probably dueto a membrane change (G. Nass and J. Thomale, FEBS Lett. 39:182-186(1974)).

[0033] None of these mutant strains, however, has been used for thefermentative production of L-threonine.

[0034]E. coli strains have recently been described which containchromosomally integrated thr operons under the regulatory control of anon-native promoter (Wang et al., U.S. Pat. No. 5,939,307, the entiredisclosure of which is incorporated herein by reference). One of thesestrains, ADM Kat 13, was shown to produce as much as 102 g/L ofL-threonine after 48 hours in culture.

[0035] There remains a need in the art for microorganism strains whichare readily culturable and efficiently produce large amounts of aminoacids such as threonine and isoleucine.

SUMMARY OF THE INVENTION

[0036] One object of the present invention is to provide microorganismswhich efficiently produce amino acids (e.g., L-threonine) in largeamounts and high yields. In general, microorganisms of the invention donot require any recombinant plasmids containing genes that encodeenzymes in the biosynthesis of the amino acid product and, in mostinstances, have no amino acid nutritional requirements.

[0037] When bacterial strains of the invention over-produce L-threonine,in many instances, these strains will be resistant to either L-threonineitself or threonine raffinate (TRF).

[0038] In one embodiment, the invention is directed to processes forproducing Escherichia coli strains capable of producing between about 95and about 150 g/L of L-threonine by about 48 hours of growth in culturecomprising:

[0039] (a) inserting into the chromosome of an E. coli at least onethreonine operon operably linked to a non-native promoter to produce aparent strain; and

[0040] (b) performing at least one cycle of mutagenesis on the parentstrain, followed by screening the mutagenized cells to identify E. coliwhich produce between about 95 and about 150 g/L of L-threonine by about48 hours of growth in culture. The invention also includes E. colistrains produced by the above processes.

[0041] In related embodiments, the invention is directed to processesfor producing E. coli strains capable of producing between about 110 andabout 120 g/L of L-threonine, between about 110 and about 130 g/L ofL-threonine, or between about 100 and about 140 g/L of L-threonine byabout 48 hours of growth in culture.

[0042] In additional related embodiments, the invention is directed toprocesses for producing E. coli strains employing agents such asalkylating agents, intercalating agents, and ultraviolet light to inducemutations.

[0043] In other related embodiments, the invention is directed toprocesses for producing E. coli strains having two or three threonineoperons inserted into the chromosome of the E. coli. Further, theseindividual threonine operons may be operably linked to at least twodifferent non-native promoters. Non-native promoters suitable for use inthe invention include the tac promoter, the lac promoter, the trppromoter, the ipp promoter, the P_(L) promoter, and the P_(R) promoter.

[0044] Related embodiments also include processes for producing E. colistrains having threonine operons containing genes that encodefeedback-resistant aspartate kinase-homoserine dehydrogenases. Further,E. coli strains used to generate strains of the invention may contain adefective threonine dehydrogenase gene on their chromosomes.

[0045] Strains which maybe used in the processes discussed above includethose which contain a threonine operon obtained from the E. coli straindeposited as ATCC Deposit No. 21277.

[0046] The processes described above may also be used to generatestrains which are resistant to threonine raffinate, resistant toborrelidin or cyclopentanecarboxylic acid (CPCA), or resistant to anycombination of threonine raffinate, borrelidin and CPCA. Thus, theinvention also includes strains of E. coli produced by the above processwhich are resistant to threonine raffinate, resistant to borrelidin orCPCA, or resistant to any combination of threonine raffinate, borrelidinand CPCA.

[0047] In other embodiments the invention is directed to E. coli strainscomprising at least one chromosomally integrated threonine operonoperably linked to a non-native promoter. These strains are capable ofproducing between about 110 and about 120 g/L of L-threonine, betweenabout 110 and about 130 g/L of L-threonine, between about 100 and about140 g/L of L-threonine, or between about 95 and about 150 g/L ofL-threonine by about 48 hours of growth in culture. Strains of theinvention will generally not include E. coli strains KY10935, ADM TH1.2,and ADM Kat13.

[0048] In related embodiments, the invention includes E. coli strainswhich have the above characteristics and comprise a threonine operonobtained from the E. coli strain deposited as ATCC Deposit No. 21277.

[0049] The invention also includes E. coli strains which are resistantto threonine raffinate and are capable of producing between about 110and about 120 g/L of L-threonine, between about 110 and about 130 g/L ofL-threonine, between about 100 and about 140 g/L of L-threonine, orbetween about 95 and about 150 g/L of L-threonine by about 48 hours ofgrowth in culture.

[0050] In other embodiments, the threonine operon of E. coli strains ofthe invention encodes a feedback-resistant aspartate kinase 1-homoserinedehydrogenase I gene (thrA), a homoserine kinase (thrB) gene, and athreonine synthase gene (thrC).

[0051] In further embodiments, E. coli strains of the invention containa defective threonine dehydrogenase gene on their chromosomes.

[0052] The invention also includes E. coli strains which have thecharacteristics of the strain deposited as NRRL B-30319 (AgricultureResearch Culture Collection (NRRL), 1815 N. University Street, Peoria,Ill., 61604, USA).

[0053] The invention further includes the E. coli strains deposited asNRRL B-30316, NRRL B-30317, NRRL B-30318, and NRRL B-30319 (AgricultureResearch Culture Collection (NRRL), 1815 N. University Street, Peoria,Ill., 61604, USA).

[0054] Additionally, the invention is directed to processes forproducing L-threonine comprising the steps of culturing the strainsmentioned above and recovering L-threonine produced.

BRIEF DESCRIPTION OF THE FIGURES

[0055]FIG. 1 depicts the construction of plasmid pAD 103 from Kohara'slambda 676 and plasmid pUC 19.

[0056]FIG. 2 depicts the construction of plasmid pAD106 from plasmid pAD103 and plasmid pUC4k.

[0057]FIG. 3 depicts the construction of plasmid pAD115 from plasmidpAD103 and plasmid pkk223-3.

[0058]FIG. 4 depicts the construction of plasmid pAD123 from plasmid pAD115 and plasmid pAD106.

[0059]FIG. 5 depicts the integration of the promoter region from plasmidpAD123 into the chromosome of E. coli.

[0060]FIG. 6 depicts the construction of plasmid pAD132 by the insertionof the tdh::cat deletion from E. coli strain SP942 into plasmid pUC18 .

[0061]FIG. 7 depicts the construction of plasmid pAD133 by the insertionof nucleic acid containing a kanamycin resistance gene and a thr operonoperably linked to a tac promoter into plasmid pAD132.

[0062]FIG. 8 depicts one specific embodiment of the stepwise mutagenicprocess described in Example 6 to generate strains of the inventionwhich demonstrate improved production of L-threonine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] The present invention provides strains of novel microorganismswhich, when grown in culture, produce relatively large amounts of aminoacids (e.g., L-threonine and L-isoleucine). Further provided are methodsfor producing the aforementioned strains and methods for producing aminoacids (e.g., L-threonine and L-isoleucine) using these strains. Thus,the invention is directed, in part, to novel bacterial strains which maybe used in fermentation processes for the production of amino acid suchas L-threonine or L-isoleucine.

[0064] In one aspect, the invention provides bacterial strains (e.g.,strains of E. coli) which demonstrate both resistance to raffinate andimproved growth properties. These bacterial strains allow for theproduction of amino acids in high amounts and yields.

[0065] A number of alterations can be made to bacterial cells whichalter their metabolism and confer upon them the ability to produceincreased quantities of amino acids and other metabolic products.Examples of such alterations include the following:

[0066] 1. Eliminating or reducing feedback control mechanisms of one ormore biosynthetic pathways which lead to the production of amino acidsor amino acid precursors.

[0067] 2. The enhancement of metabolic flow by either amplifying orincreasing the expression of genes which encode rate-limiting enzymes ofbiosynthetic pathways that lead to the production of amino acids oramino acid precursors.

[0068] 3. Inhibiting degradation of a desired amino acid end product orone or more intermediates and/or precursors of the desired amino acidend product.

[0069] 4. Increasing the production of amino acid intermediates and/orand precursors.

[0070] 5. When the pathway which leads to production of a desired aminoacid end product is branched, inhibiting branches which do not lead tothe amino acid to increase intermediate and/or precursor availability.

[0071] 6. Altering membrane permeability to optimize uptake of energymolecules (e.g., glucose), intermediates and/or precursors.

[0072] 7. Altering membrane permeability to optimize amino acid endproduct excretion.

[0073] 8. The enhancement of growth tolerance to relatively highconcentrations of end products (e.g., amino acids), metabolic wasteproducts (e.g., acetic acid), or metabolic side products (e.g., aminoacid derivatives either formed by the bacterial themselves or formed inthe culture medium) which are inhibitory to bacterial cell growth.

[0074] 9. The enhancement of resistance to high osmotic pressure duringculturing resulting from high concentrations of carbon sources (e.g.glucose) or end products (e.g., amino acids).

[0075] 10. The enhancement of growth tolerance to changes inenvironmental conditions (e.g., pressure, temperature, pH, etc.).

[0076] 11. Increasing activities of enzymes involved in the uptake anduse of carbon sources in the culture medium (e.g., raffinose, stachyoseor proteins, as well as other components of corn steep liquor).

[0077] Bacteria optimized for production of a particular end product(e.g., L-threonine) will generally differ from wild-type bacteria byhaving multiple characteristics (e.g., two of more characteristics setout in the list above) which lead to increased production of the desiredend product. The invention thus includes methods for producing bacterialstrains which exhibit properties set out above and produce increasedamounts of amino acids as compared to wild-type strains. The inventionalso includes bacterial strains produced by the methods disclosedherein.

[0078] I. Definitions

[0079] The following definitions are provided to clarify the subjectmatter which the inventors consider to be the present invention.

[0080] As used herein, the term “yield” refers to the amount of aproduct produced in relation to the amount of a starting material. Withrespect to amino acids produced by a microorganism, yield refers to theamount of amino acid produced with respect to the amount ofintermediate, precursor or nutrient provided. For example, when 100grams of dextrose is supplied to a microorganism which produces 25 gramsof L-isoleucine, the yield of L-isoleucine, with respect to thedextrose, is 25%.

[0081] As used herein, the term “raffinate” refers to wastestreamproducts generated from ion-exchange operations for amino acid recoveryfrom fermentation broth in which bacteria have been cultured.

[0082] As used herein, the phrase “threonine raffinate (TRF)” refers towastestream products generated from ion-exchange operations forthreonine recovery from fermentation broth in which bacteria thatproduce threonine have been cultured.

[0083] As one skilled in the art would recognize, TRF is a heterogenouscomposition, the content of which will vary with a number of factors(e.g., the composition of the initial culture medium, the nutritionalrequirements of the cultivated organism(s), metabolic products producedby the cultivated organism(s), and chromatographic preparation processused). For purposes of selecting and identifying bacteria which areresistant to TRF, TRF will generally have the characteristics set outherein in Section II.

[0084] As used herein, the term “strain” refers to bacteria of aparticular species which have common characteristics. Unless indicatedto the contrary, the terms “strain” and “cell” are used interchangeablyherein. As one skilled in the art would recognize, bacterial strains arecomposed of individual bacterial cells. Further, individual bacterialcells have specific characteristics (e.g., a particular level ofresistance to TRF) which identifies them as being members of theirparticular strain.

[0085] As used herein, the term “mutation” refers to an insertion,deletion or substitution in a nucleic acid molecule. When present in thecoding region of a nucleic acid, a mutation may be “silent” (i.e.,results in no phenotypic effect) or may alter the function of theexpression product of the coding region. When a mutation occurs to theregulatory region of a gene or operon, the mutation may either have noeffect or alter the expression characteristics of the regulated nucleicacid.

[0086] As used herein, the term “mutagenesis” refers to a processwhereby one or more mutations are generated in an organism's geneticmaterial (e.g., DNA). With “random” mutagenesis, the exact site ofmutation is not predictable, occurring anywhere in the chromosome of themicroorganism. Further, with random mutagenesis, the mutations aregenerally brought about as a result of physical damage to the organism'snucleic acid caused by agents such as radiation or chemical treatment.As discussed in more detail below, numerous agents may be used toperform mutagenesis.

[0087] As used herein, the phrase “cycle of mutagenesis” in generalrefers to the treatment of cells with a mutagen, or combination ofmutagens, followed by culture of those cells to allow surviving cells toreproduce. In many instances, the mutagenized cells will be screened toidentify those with particular characteristics after each cycle ofmutagenesis. Further, as part of a cycle of mutagenesis, cells treatedwith a mutagen may be exposed to a selective agent (e.g., TRF)immediately after mutagenesis or while still exposed to the mutagen.

[0088] As used herein, the term “phenotype” refers to observablephysical characteristics dependent upon the genetic constitution of amicroorganism. Examples of phenotypes include the ability to expressparticular gene products and the ability to produce certain amounts of aparticular amino acid in a specified amount of time.

[0089] As used herein, the term “over-produce” refers to the productionof a compound by a cell in an amount greater than the amount produced bya reference strain (e.g., a parent strain). One example of anover-producing strain would be a strain generated from a parent strain(i.e., the reference strain) using mutagenesis which produces moreL-threonine than the parent. Thus, the strain generated by mutagenesiswould “over-produce” L-threonine in comparison to the parent, referencestrain.

[0090] As used herein, the term “operon” refers to a unit of bacterialgene expression and regulation. Operons are generally composed ofregulatory elements and at least one open reading frame (ORF). Anexample of an operon is the threonine operon of E. coli which iscomposed of a regulatory region and three open reading frames. Anotherexample of an operon is the isoleucine operon ofE. coli which iscomposed of a regulatory region and four open reading frames.

[0091] As used herein, the term “parent strain” refers to a strain of amicroorganism subjected to mutagenesis to generate a microorganism ofthe invention. Thus, use of the phrase “parent strain” does notnecessarily equate with the phrase “wild-type” or provide informationabout the history of the referred to strain.

[0092] II. Strains of the Invention and Their Preparation

[0093] Novel bacterial strains of the present invention have thefollowing characteristics:

[0094] (1) they contain at least one operon which (a) is integrated intothe bacterial chromosome, (b) is under the control of a non-nativepromoter, and (c) encodes enzymes involved in amino acid synthesis; and

[0095] (2) they are capable of producing one or more amino acids upongrowth in culture.

[0096] In particular embodiments, novel bacterial strains of the presentinvention include strains which have the following characteristics:

[0097] (1) they contain at least one thr operon (i.e., contain at leastone set of the genes encoding threonine biosynthetic enzymes) which (a)is integrated into the bacterial chromosome and (b) is under the controlof a non-native promoter; and

[0098] (2) they are capable of producing either L-threonine orL-isoleucine upon growth in culture.

[0099] A. Operons Suitable for Use With the Invention

[0100] While, as explained below, the invention can be used to producecells which over-produce a considerable number of amino acids, theinvention is discussed below mainly with respect to cells whichover-produce L-threonine and L-isoleucine, as well as processes forproducing these amino acids.

[0101] The threonine (thr) operon on the chromosome of cells ofbacterial strains included within the scope of the invention encodesenzymes necessary for threonine biosynthesis. Due to the fact thatseveral enzymes are capable of catalyzing reactions to produce variousintermediates in the threonine pathway, the genes present in thethreonine operon employed can vary. For example, the threonine operoncan be composed of an AK-HD gene (thrA or metL), a homoserine kinasegene (thrB), and a threonine synthase gene (thrC). Further, the throperon can be composed of thrA (the AK I-HD I gene), thrB and thrc.Suitable thr operons maybe obtained, for example, from E. coli strainsdeposited with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, USA and assigned ATCCDeposit Nos. 21277 and 21530.

[0102] Further, multiple copies of the thr operon may be present on thechromosomes of bacterial cells of the invention. Increased copy numberof the thr operon will generally result in increased expression of thegenes of this operon upon induction.

[0103] In many instances, the thr operon contains at least onenon-attenuated gene (i.e., expression of the gene is not suppressed bythe levels (extra- and/or intra-cellular) of one or more of thethreonine biosynthetic enzymes and/or the products thereof (e.g.,L-threonine and L-isoleucine)). The inventive strains may also contain athr operon having a defective thr attenuator (the regulatory regiondownstream of the transcription initiation site and upstream of thefirst structural gene) or a thr operon that lacks the thr attenuatoraltogether.

[0104] In one specific embodiment, the thr operon encodes one or morefeedback-resistant threonine biosynthetic enzymes (e.g., the activity ofthe enzyme is not inhibited by the extra- and/or intra-cellular levelsof the intermediates and/or products of threonine biosynthesis). In amore specific embodiment, the thr operon contains a gene that encodes afeedback-resistant AK-HD, such as a feedback-resistant AK I-HD I. Use ofa feedback-resistant AK-HD provides a higher level of enzymatic activityfor threonine biosynthesis, even in the presence of the L-threoninebeing produced.

[0105] Expression of the threonine operon(s) in strains of the inventionwill generally be controlled by a non-native promoter (i.e., a promoterthat does not control expression of the thr operon in bacterial strainsnormally found in nature). Replacing the native promoter of thethreonine biosynthetic enzymes with a strong non-native promoter tocontrol expression of the thr operon results in higher threonineproduction even with only a single, genomic copy of the thr operon. Inaddition, when a non-native promoter is used to control expression ofthreonine operon, it is not necessary to render the bacterial strainsauxotrophic for isoleucine to achieve this higher threonine production.Illustrative examples of promoters suitable for use in E. coli include,but are not limited to: the lac promoter, the trp promoter, the P_(L)promoter of λ bacteriophage, the P_(R) promoter, the /pp promoter, andthe tac promoter. In one specific embodiment, the tac promoter is used.

[0106] In addition to the threonine operon, cells of the inventivebacterial strains may also contains at least one gene encoding aspartatesemialdehyde dehydrogenase (asd) either integrated into theirchromosomes or present on an extrachromosomal element (e.g., a plasmid).For example, the chromosome in cells of the present invention maycontain at least one asd gene, at least one thrA gene, at least one thrBgene and/or at least one thrc gene. Of course, one, two, three, or morecopies of each of these genes may be present.

[0107] Threonine dehydrogenase (tdh) catalyzes the oxidation ofL-threonine to a-amino-β-ketobutyrate. Accordingly, in one specificembodiment, the chromosome of the inventive cells further contains atleast one defective threonine dehydrogenase (tdh⁻) gene. The defectivetdh gene may be a gene having a reduced level of expression of threoninedehydrogenase or a gene that encodes a threonine dehydrogenase mutanthaving reduced enzymatic activity relative to that of native threoninedehydrogenase. For example, the defective tdh gene employed in inventivestrains does not express threonine dehydrogenase. Illustrative examplesof suitable tdh⁻ genes that do not express threonine dehydrogenaseinclude a tdh gene having a chloramphenicol acetyltransferase (cat) geneinserted into it or a tdh gene having transposon Tn5 inserted into it,as described in U.S. Pat. No. 5,175,107.

[0108] The invention further provides microorganisms which expressincreased amounts of enzymes which catalyze the production ofL-isoleucine, as well as microorganisms which over-produce L-isoleucine.As one skilled in the art, bacterial strains of the invention whichproduce increased quantities of L-threonine, in effect, allow for theproduction of substantial quantities of L-isoleucine. This is sobecause, as already discussed, L-threonine is a precursor ofL-isoleucine. Thus, operons suitable for use with the present inventioninclude the isoleucine operon of E. coli, which is composed of the ilvA,ilvGM, ilvD, and ilvE genes.

[0109] In one embodiment of the invention, an isoleucine operon underthe control of a non-native promoter is introduced into microorganisms.Further, nucleic acid encoding dihydroxyacid reductoisomerase (ilvC) mayalso be introduced into cells. These genes maybe either inserted intochromosomal DNA or carried on plasmids.

[0110] In addition, because the reactions catalyzed by threoninedeaminase and aceto-hydroxyacid synthetase are believed to be the ratelimiting steps in the production of isoleucine, it will be advantages,when the production of isoleucine is desired, to over-express theseparticular gene products.

[0111] In addition, because the gene products of the ilvA gene (i.e.,threonine deaminase) and the ilvGM (i.e., aceto-hydroxyacid synthetaseII) are inhibited, respectively, by L-isoleucine and L-valine, in manycircumstances, it will generally be advantageous to use feed-backresistant forms of these enzymes.

[0112] Similar modifications of cells and process of the invention canbe readily employed to produce other amino acids generated by pathwaysrelated to those for the production of L-threonine and L-isoleucine.Examples of amino acids which can be produced using such modificationsinclude L-lysine and L-glycine.

[0113] The invention also provides microorganisms which expressincreased amounts of enzymes which catalyze the production ofL-methionine, as well as microorganisms which over-produce L-methionine.Examples of such microorganisms are ones which contain at least one metoperon on the chromosome (i.e., the metL gene (which encodes AK II-HDII), the metA gene (homoserine succinyltransferase), the metB gene(cystathionine β-synthase), the metC gene (cystathionine β-lyase) andthe metE and metH genes (homocysteine methylase)) that have beensubjected to mutagenesis and screening steps described herein. The genesset out in the preceding sentence, including feedback-resistant variantsthereof, and, optionally, a non-native promoter can be introduced intothe chromosome of the host microorganism according to one or more of thegeneral methods discussed herein and/or known to those skilled in theart.

[0114] As indicated above, microorganisms which over-produce lysine canalso be prepared by subjecting microorganisms that contain genesencoding lysine biosynthetic enzymes (e.g., a feedback-resistant lysinebiosynthetic enzyme encoded by lysC and/or dapA) and, optionally, anon-native promoter to mutagenesis and screening steps described herein.

[0115] Bacterial strains of the present invention may be prepared by anyof the methods and techniques known and available to those skilled inthe art. Illustrative examples of suitable methods for constructing theinventive bacterial strains include gene integration techniques (e.g.,mediated by transforming linear DNA fragments and homologousrecombination) and transduction mediated by the bacteriophage P1. Thesemethods are well known in the art and are described, for example, in J.H. Miller, Experiments in Molecular Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A ShortCourse in Bacterial Genetics, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1992); M. Singer and P. Berg, Genes & Genomes,University Science Books, Mill Valley, Calif. (1991); J. Sambrook, E. F.Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); P.B. Kaufman et al., Handbook of Molecular and Cellular Methods in Biologyand Medicine, CRC Press, Boca Raton, Fla. (1995); Methods in PlantMolecular Biology and Biotechnology, B. R. Glick and J. E. Thompson,eds., CRC Press, Boca Raton, Fla. (1993); and P. F. Smith-Keary,Molecular Genetics of Escherichia coli, The Guilford Press, New York,N.Y. (1989), the entire disclosure of each of which is incorporatedherein by reference.

[0116] B. Amino Acid Production

[0117] Bacterial strains of the present invention include strains whichare capable of producing substantial quantities of L-threonine orL-isoleucine when grown in culture. In particular, when grown inculture, strains of the invention include strains which are capable ofproducing at least about 65 g/L of L-threonine in about 36 hours, atleast about 75 g/L of L-threonine in about 36 hours, at least about 85g/L of L-threonine in about 36 hours, at least about 95 g/L ofL-threonine in about 36 hours, at least about 105 g/L of L-threonine inabout 36 hours, at least about 110 g/L of L-threonine in about 36 hours,at least about 115 g/L of L-threonine in about 36 hours, at least about120 g/L of L-threonine in about 36 hours, at least about 125 g/L ofL-threonine in about 36 hours, at least about 130 g/L of L-threonine inabout 36 hours, at least about 135 g/L of L-threonine in about 36 hours,at least about 140 g/L of L-threonine in about 36 hours, at least about145 g/L of L-threonine in about 36 hours, or at least about 150 g/L ofL-threonine in about 36 hours. Further, the inventive strains includestrains which are capable of producing at least about 95 g/L ofL-threonine in about 48 hours, at least about 100 g/L of L-threonine inabout 48 hours, at least about 105 g/L of L-threonine in about 48 hours,at least about 110 g/L of L-threonine in about 48 hours, at least about115 g/L of L-threonine in about 48 hours, at least about 120 g/L ofthreonine in about 48 hours, at least about 125 g/L of L-threonine inabout 48 hours, at least about 130 g/L of L-threonine in about 48 hours,at least about 135 g/L of L-threonine in about 48 hours, at least about140 g/L of L-threonine in about 48 hours, at least about 145 g/L ofL-threonine in about 48 hours, or at least about 150 g/L of threonine inabout 48 hours.

[0118] Further, the inventive strains include strains which are capableof producing L-threonine at a rate of at least about 2 g/L/hr, at leastabout 2.5 g/L/hr, at least about 3 g/L/hr, at least about 3.6 g/L/hr, atleast about 4.0 g/L/hr, at least about 4.5 g/L/hr, or at least about 5.0g/L/hr.

[0119] In addition, when grown in culture, the inventive strains includestrains which are capable of producing between about 75 and about 95 g/Lof L-threonine in about 36 hours, between about 80 and about 100 g/L ofL-threonine in about 36 hours, between about 85 and about 105 g/L ofL-threonine in about 36 hours, between about 90 and about 110 g/L ofL-threonine in about 36 hours, between about 95 and about 110 g/L ofL-threonine in about 36 hours, between about 100 and about 115 g/L ofL-threonine in about 36 hours, between about 100 and about 120 g/L ofL-threonine in about 36 hours, between about 100 and about 125 g/L ofL-threonine in about 36 hours, between about 100 and about 130 g/L ofL-threonine in about 36 hours, between about 100 and about 135 g/L ofL-threonine in about 36 hours, between about 100 and about 140 g/L ofL-threonine in about 36 hours, between about 105 and about 120 g/L ofL-threonine in about 36 hours, between about 110 and about 120 g/L ofL-threonine in about 36 hours, between about 110 and about 125 g/L ofL-threonine in about 36 hours, between about 110 and about 130 g/L ofL-threonine in about 36 hours, between about 110 and about 135 g/L ofL-threonine in about 36 hours, between about 110 and about 140 g/L ofL-threonine in about 36 hours, between about 115 and about 130 g/L ofL-threonine in about 36 hours, between about 120 and about 135 g/L ofL-threonine in about 36 hours, between about 95 and about 135 g/L ofL-threonine in about 36 hours, between about 95 and about 120 g/L ofL-threonine in about 36 hours, between about 95 an d about 125 g/L of Lthreonine in about 36 hours, between about 95 and about 135 g/L ofL-threonine in about 36 hours, between about 95 and about 14 5 g/L ofL-threonine in about 36 hours, between about 95 and about 150 g/L ofL-threonine in about 36 hours, between about 105 and about 125 g/L ofL-threonine in about 36 hours, between about 105 and about 130 g/L ofL-threonine in about 36 hours, or between about 105 and about 135 g/L ofL-threonine in about 36 hours.

[0120] Further, when grown in culture, the inventive strains includestrains which are capable of producing between about 80 and about 100g/L of L-threonine in about 48 hours, between about 85 and about 105 g/Lof L-threonine in about 48 hours, between about 90 and about 110 g/L ofL-threonine in about 48 hours, between about 95 and about 110 g/L ofL-threonine in about 48 hours, between about 100 and about 115 g/L ofL-threonine in about 48 hours, between about 105 and about 120 g/L ofL-threonine in about 48 hours, between about 110 and about 125 g/L ofL-threonine in about 48 hours, between about 115 and about 130 g/L ofL-threonine in about 48 hours, between about 120 and about 135 g/L ofL-threonine in about 48 hours, between about 125 and about 140 g/L ofL-threonine in about 48 hours, between about 95 and about 115 g/L ofL-threonine in about 48 hours, between about 95 and about 120 g/L ofL-threonine in about 48 hours, between about 95 and about 125 g/L ofL-threonine in about 48 hours, between about 95 and about 135 g/L ofL-threonine in about 48 hours, between about 95 and about 145 g/L ofL-threonine in about 48 hours, between about 95 and about 150 g/L ofL-threonine in about 48 hours, between about 100 and about 120 g/L ofL-threonine in about 48 hours, between about 100 and about 125 g/L ofL-threonine in about 48 hours, between about 100 and about 130 g/L ofL-threonine in about 48 hours, between about 100 and about 135 g/L ofL-threonine in about 48 hours, between about 100 and about 140 g/L ofL-threonine in about 48 hours, between about 100 and about 145 g/L ofL-threonine in about 48 hours, between about 105 and about 125 g/L ofL-threonine in about 48 hours , between about 105 and about 130 g/L ofL-threonine in about 48 hours, between about 105 and about 135 g/L ofL-threonine in about 48 hours, between about 105 and about 140 g/L ofL-threonine in about 48 hours, between about 105 and about 145 g/L ofL-threonine in about 48 hours, between about 105 and about 150 g/L ofL-threonine in about 48 hours, between about 110 and about 120 g/L ofL-threonine in about 48 hours, between about 110 and about 130 g/L ofL-threonine in about 48 hours, between about 110 and about 135 g/L ofL-threonine in about 48 hours, between about 110 and about 140 g/L ofL-threonine in about 48 hours, between about 115 and about 125 g/L ofL-threonine in about 48 hours, between about 115 and about 13 5 g/L ofL-threonine in about 48 hours, between about 115 and about 140 g/L ofL-threonine in about 48 hours, between about 115 and about 145 g/L ofL-threonine in about 48 hours, or between about 115 and about 150 g/L ofL-threonine in about 48 hours.

[0121] The bacterial strains of the invention also include strains whichproduce L-threonine in high yield with respect to the carbon sourcepresent in the culture medium. Thus, the strains of the inventioninclude strains which, with reference to the dextrose content of theculture medium, produce L-threonine in the following yields (wt./wt.):about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%,about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about45%, about 46%, about 47%, about 48%, about 49%, or about 50%.

[0122] Strains of the invention include strains which, with reference tothe dextrose content of the culture medium, produce L-threonine in thefollowing ranges of yields (wt./wt.): between about 25% and about 40%,between about 30% and about 35%, between about 30% and about 45%,between about 30% and about 50%, between about 35% and about 40%,between about 35% and about 45%, between about 35% and about 50%,between about 40% and about 45%, and between about 40% and about 50%.

[0123] Strains of the invention include strains which are capable ofproducing at least about 65 g/L of L-isoleucine in about 36 hours, atleast about 75 g/L of L-isoleucine in about 36 hours, at least about 85g/L of L-isoleucine in about 36 hours, at least about 95 g/L ofL-isoleucine in about 36 hours, at least about 105 g/L of L-isoleucinein about 36 hours, at least about 115 g/L of L-isoleucine in about 36hours, at least about 125 g/L of L-isoleucine in about 36 hours, atleast about 130 g/L of L-isoleucine in about 36 hours, at least about135 g/L of L-isoleucine in about 36 hours, or at least about 140 g/L ofL-isoleucine in about 36 hours. Further, the inventive strains includestrains which are capable of producing at least about 90 g/L ofL-isoleucine in about 48 hours, at least about 100 g/L of L-isoleucinein about 48 hours, at least about 110 g/L of L-isoleucine in about 48hours, at least about 120 g/L of L-isoleucine in about 48 hours, atleast about 130 g/L of L-isoleucine in about 48 hours, at least about140 g/L of L-isoleucine in about 48 hours, or at least about 150 g/L ofL-isoleucine in about 48 hours.

[0124] Further, the inventive strains include strains which are capableof producing L-isoleucine at a rate of at least about 2 g/L/hr, at leastabout 2.5 g/L/hr, at least about 3 g/L/hr, at least about 3.6 g/L/hr, atleast about 4.0 g/L/hr, at least about 4.5 g/L/hr, or at least about 5.0g/L/hr.

[0125] In addition, when grown in culture, the inventive strains includestrains which are capable of producing between about 75 and about 95 g/Lof L-isoleucine in about 36 hours, between about 85 and about 105 g/L ofL-isoleucine in about 36 hours, between about 95 and about 115 g/L ofL-isoleucine in about 36 hours, between about 105 and about 125 g/L ofL-isoleucine in about 36 hours, between about 115 and about 135 g/L ofL-isoleucine in about 36 hours, or between about 125 and about 145 g/Lof L-isoleucine in about 36 hours.

[0126] Further, when grown in culture, the inventive strains includestrains which are capable of producing between about 80 and about 100g/L of L-isoleucine in about 48 hours, between about 85 and about 105g/L of L-isoleucine in about 48 hours, between about 90 and about 110g/L of L-isoleucine in about 48 hours, between about 100 and about 120gL of L-isoleucine in about 48 hours, between about 110 and about 130g/L of L-isoleucine in about 48 hours, between about 120 and about 140g/L of L-isoleucine in about 48 hours, or between about 130 and about150 g/L of L-isoleucine in about 48 hours.

[0127] The bacterial strains of the invention also include strains whichproduce L-isoleucine in high yield with respect to the carbon sourcepresent in the culture medium. Thus, the strains of the inventioninclude strains which, with reference to the dextrose content of theculture medium, produce L-isoleucine in the following yields (wt./wt.):about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%,about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about45%, about 46%, about 47%, about 48%, about 49%, or about 50%.

[0128] Strains of the invention include strains which, with reference tothe dextrose content of the culture medium, produce L-isoleucine in thefollowing ranges of yields (wt./wt.): between about 25% and about 40%,between about 30% and about 35%, between about 30% and about 45%,between about 30% and about 50%, between about 35% and about 40%,between about 35% and about 45%, between about 35% and about 50%,between about 40% and about 45%, and between about 40% and about 50%.

[0129] The amount of L-threonine or L-isoleucine, as well as other aminoacids, present in culture media can be measured by a number of methods.For example, as indicated below in Examples 2 and 5, the amount ofL-threonine, as well as other amino acids, present in culture media canbe determined using HPLC. L-threonine or L-isoleucine levels can also bedetermined using methods such as paper chromatography with ninhydrindetection, thin layer chromatography, or microbiological assay.

[0130] C. Preparation of Bacterial Strains Capable of Over-ProducingAmino Acids

[0131] As discussed above, bacterial strains well suited for commercialproduction of amino acids will generally be altered in more than onephenotypic trait related to production/excretion of the particular aminoacids as compared to wild-type strains. As also discussed above,bacterial strains of the invention which over-produce amino acidsinclude strains which contain at least one thr operon which (a) isintegrated into the bacterial chromosome the chromosome and (b) is undercontrol of a non-native promoter. These strains will also generallycontain phenotypic changes related to one, two, three, four, or more ofthe following: (1) the elimination or reduction of feed-back controlmechanisms for one, two, three or more biosynthetic pathways which leadto production of amino acids or amino acid precursors; (2) theenhancement of metabolic flow by either amplification or increasingexpression of genes which encode rate-limiting enzymes of biosyntheticpathways which lead to the production of amino acids (e.g., L-threonineor L-isoleucine) or amino acid precursors (e.g., aspartate); (3) theinhibition of degradation pathways involving either the desired aminoacid end product (e.g., L-threonine or L-isoleucine), intermediates(e.g., homoserine), and/or precursors (e.g., aspartate); (4) increasedproduction of intermediates and/or and precursors; (5) when the pathwaywhich leads to production of a desired amino acid end product isbranched, inhibition of branches which do not lead the desired endproduct or an intermediate and/or a precursor of the desired end product(e.g., inhibiting the E. coli methionine pathway, when the desired endproduct is L-threonine or L-isoleucine); (6) alterations in membranepermeability to optimize uptake of energy molecules (e.g., glucose),intermediates and/or precursors; (7) alterations in membranepermeability to optimize amino acid end product (e.g., L-threonine orL-isoleucine) excretion; (8) the enhancement of growth tolerance torelatively high concentrations of end products (e.g., amino acids),metabolic waste products (e.g., acetic acid), or metabolic side products(e.g., amino acid derivatives) which are inhibitory to bacterial cellgrowth; (9) the enhancement of resistance to high osmotic pressureduring cultivation resulting from increased concentrations of carbonsources (e.g., glucose) or end products (e.g., amino acids); (10) theenhancement of growth tolerance to changes in environmental conditions(e.g., pressure, temperature, pH, etc.); and (11) increasing activitiesof enzymes involved in the uptake and use of carbon sources in theculture medium (e.g., raffinose, stachyose or proteins, as well as othercomponents of corn steep liquor).

[0132] The invention also includes methods for screening bacterial cellsto identify cells which have been subjected to mutagenesis and have one,two, three, four, or more of the characteristics set out above. Furtherincluded in the invention are bacterial strains which have one, two,three, four, or more of the above characteristics.

[0133] As one skilled in the art would recognize, the use of randommutagenesis, followed by screening to identify cells of the inventionwhich over-produce a desired amino acid (e.g., L-threonine orL-isoleucine) results in the selection of cells having phenotypicchanges that do not necessarily provide an indication of the mechanismby which the cell over-produces the amino acid. For example, amino acidover-production could be related to pleiotropic effect of an apparentunrelated phenotypic alteration. Thus, the invention is not limited tocells which over-produce amino acids and exhibit one or more of themetabolic alterations set out in the preceding list. In other words, theinvention includes cells which are characterized by the ability toproduce specified quantities of particular amino acids upon growth inculture for specified periods of time.

[0134] In specific embodiments, the strains of the invention areproduced by subjecting bacterial cells containing at least one throperon on the chromosome under the control of a non-native promoter toone, two, three, four, five, or more cycles of mutagenesis followed byscreening to identify cells demonstrating increased production of aminoacids (e.g., L-threonine or L-isoleucine).

[0135] A considerable number of methods for performing metagenesis areknown in the art and can be used to generate bacterial strains of theinvention. In general, these methods involve the use of chemical agentsor radiation for inducing mutations.

[0136] Examples of classes of chemical compound used in mutagenicprocedures are alkylating and ethylating agents, such asN-methyl-N-nitrosourea N-nitroso-N,N-diethylamine (NDEA) andN-ethyl-N′-nitro-N-nitrosoguanidine (ENNG), which have been known forsome time to induce mutations in nucleic acid molecules (Hince et al.,Mutat. Res. 46:1-10 (1977); J. Jia et al., Mutat. Res. 352:39-45(1996)).

[0137] Intercalating agents, such as ethidium bromide, as well as otheragents which bind to nucleic acid molecules, have also been shown tohave mutagenic activity. For example, SYBR Green I stain, anon-intercalating nucleic acid stain, has been shown using the Ames testto induce mutations (Singer et al., Mutat.

[0138] Res. 439:37-47 (1999)).

[0139] Other agents which can be used to induce mutations includehydroxylamine, bisulfites, nitrofurans (e.g., 7-methoxy-2-nitronaphtho[2,1-β] furan (R7000)), and agents which induce oxidative stress (P.Quillardet et al., Mutat. Res. 358:113-122 (1996); G. Wang et al., MolGen. Genet. 251:573-579 (1996)).

[0140] One skilled in the art would understand how to adjust theconcentrations of the mutagenic agent and/or the particular conditionsto achieve a desired mutation rate. For example, when ionizing radiationis used to produce mutagenized cells, the intensity of the radiation orduration of exposure can be adjusted to induce a particular number ofmutations per cell. Further, the intensity of the radiation or durationof exposure can also be adjusted so that a particular percentage (e.g.,5%) of the treated cells survive.

[0141] After cells have been subjected to mutagenesis, they can bescreened to determine whether they have particular characteristics. Itis noted long these lines that a number of characteristics have beenassociated with increased production of L-threonine or L-isoleucine bybacterial cells. Example of such characteristics include resistance tocysteine, threonine, methionine, and purine analogs; resistance toisoleucine antagonists; impaired uptake of L-threonine uptake; andaltered feedback inhibition of enzymes in the threonine and isoleucinebiosynthetic pathways (see, e.g., Takano et al., U.S. Pat. No.5,087,566; Yamada et al., U.S. Pat. No. 5,098,835; Yamada et al., U.S.Pat. No. 5,264,353; Kino et al., U.S. Pat. No. 5,474,918; K. Okamoto etal., Biosci. Biotechnol. Biochem. 61:1877-1882 (1997); Sahm et al.,Annals N. Y Acad. Sci 782:25-39 (1996); Hashiguchi et al., Biosci.Biotechnol. Biochem. 63:672-679 (1999)). Other characteristics believedto correlate with increased production of L-threonine include resistanceto L-threonine and TRF.

[0142] Further, screening/selection of cells having an L-threonineresistant phenotype may be done in media containing from about 1% toabout 15% (weight/volume) L-threonine. For example, microorganisms ofthe invention can be screened using culture media containing about 1%,1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%,2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.2%, 3.4%, 3.6%, 3.8%,4%, 4.2%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, 6.2%, 6.4%,6.6%, 6.8%, 7%, 7.2%, 7.4%, 7.6%, 7.8%, 8%, 8.2%, 8.4%, 8.6%, 8.8%, 9%,9.2%, 9.4%, 9.6%, 9.8%, 10%, 10.2%, 10.4%, 10.6%, 10.8%, 11%, 11.2%,11.4%, 11.6%, 11.8%, 12%, 12.2%, 12.4%, 12.6%, 12.8%, 13%, 13.2%, 13.4%,13.6%, 13.8%, 14%, 14.2%, 14.4%, 14.6%, 14.8%, 15%, 15.2%, 15.4%, 15.6%,15.8%, or 16% L-threonine.

[0143] Strains of the invention may be generated by using multiplecycles of mutagenesis and screening. After each mutagenic treatment, themutagenized cells can be screened for either (1) increased production ofa desired amino acid end product (e.g., L-threonine or L-isoleucine) or(2) one, two, three, four, five, or more characteristics associated withincreased production of the end product (e.g., L-threonine orL-isoleucine), followed by screening for increased production of thedesired amino acid end product (e.g., L-threonine or L-isoleucine).

[0144] As noted above, one characteristic associated with increasedthreonine production is resistance to TRF. Thus, the invention includesbacterial strains which are resistant to TRF, as well as methods forproducing and identifying TRF resistant mutants.

[0145] TRF can be prepared, for example, by protocols similar to thefollowing.

[0146] Particular matter is removed by ultrafiltration from conditionedthreonine fermentation broth prepared, for example, as described belowin Example 9 using fermentor fermentation medium. The permeate is thenevaporated to concentrate threonine. Crystalized threonine is thenrecovered from the concentrated broth by centrifugation, using, forexample, a continuous flow rotor. The liquid separated from thethreonine is then processes through an ion exchange chromatographicseparation system, such as C-SEP or I-SEP (Advanced SeparationTechnologies, Inc., St. Petersburg, Fla.). The waste effluent obtainedtherefrom is referred to as a “TRF” solution.

[0147] As one skilled in the art would recognize, separation methodsother than C-SEP or I-SEP could also be employed. Ion exchangechromatographic separation systems are commonly known in the art, asexemplified in U.S. Pat. Nos. 4,808,317 and 4,764,276, which areincorporated herein by reference.

[0148] One TRF preparation prepared by the inventors was analyzed andfound to contain the following components: aspartic acid (63 ppm),threonine (438 ppm), glutamic acid (24 ppm), proline (<14 ppm), glycine(40 ppm), alanine (16 ppm), cystine (42 ppm), valine (<18 ppm),methionine (232 ppm), isoleucine (297 ppm), leucine (25 ppm), tyrosine(31 ppm), phenylalanine (22 ppm), lysine (152 ppm), serine (<1 ppm),histidine (1 ppm), arginine (<22 ppm), ammonia (1,791 ppm), raffinose(5,036 ppm), sucrose (1,885 ppm), glucose (1,344 ppm), and fructose (725ppm).

[0149] As can be seen from the above, TRF contains a considerable amountof ammonia sulfate, L-threonine, other amino acids, salts, andcarbohydrates. Thus, TRF contains nitrogen sources, such as ammoniasulfate, and nutrients, such as amino acids and carbohydrates, which canbe metabolized by microorganisms.

[0150] TRF concentration may be determined by determining theconcentration of a reference component present in the TRF. One exampleof a reference component is ammonium sulfate. Unless otherwise statedherein, the concentration of a TRF solution is based on the percentageof ammonium sulfate present (wt./vol.). For example, a 5% TRF solutionwould contain 5 grams of ammonium sulfate per 100 milliliters of solute.

[0151] The ammonium sulfate concentration of a solution can bedetermined using a number of methods. For example, an ion selectiveprobe can be used to measure the concentration of ammonium ions (e.g.,ORION Research, Inc., 500 Cummings Center, Beverly, Mass. 01915, CatalogNo. 931801).

[0152] When TRF is used to either (1) generate bacterial strains whichover-produce L-threonine or (2) identify TRF resistant bacterialstrains, the TRF will generally be prepared as described below inExample 10.

[0153] Raffinate solutions maybe sterilized by any number of means priorto use in protocols for generating and screening raffinate resistantbacterial strains. The inventors have determined that sterilization ofraffinate containing media, especially at high concentrations ofsolutes, using heat treatment produces amino acid derivatives and othermetabolic antagonists which inhibit culture growth. However, heatsterilized TRF containing medium maybe used to select mutants that areresistant to amino acid derivatives, especially L-threonine derivatives,through the improvement of their threonine production. To avoidalterations in raffinate properties associated with heat sterilization,culture media may be sterilized, for example, by ultrafiltration.

[0154] Strains of the invention include strains having an improvedraffinate resistant phenotype, which is determined by the concentrationof raffinate, as measured by ammonium sulfate content, in the selectionmedium employed. As discussed above, selection for raffinate resistantmutants maybe done in a culture media containing raffinate. Theparticular concentration of raffinate present in the selection mediumwill vary with factors such as the medium itself, the cells beingscreened for raffinate resistance, and the raffinate preparation itself.For example, TRF resistant E. coli may be selected using minimal mediumE (see Examples 6 and 7) containing from about 0.2% to about 0.5%raffinate. As one skilled in the art would understand, the TRFconcentrations used will also vary with factors such as the genus andspecies of bacteria used and the initial sensitivity of the bacterialstrain to TRF.

[0155] Bacterial strains of the invention may be made by performingmutagenesis on a parent bacterial strain followed by selection for cellsexhibiting a TRF-resistant phenotype. Parent microorganisms may beselected from any organism useful for the fermentative production ofamino acids (e.g., L-threonine); however, in most instances, theorganism will be a strain ofE. coli.

[0156] Screening/selection of cells having a TRF-resistant phenotype maybe performed in culture media containing from about 0.05% to about 5%TRF. For example, microorganisms of the invention can be screened usingculture media containing about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%,0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%,0.9%, 0.95%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%,2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%,4.4%, 4.6%, 4.8%, or 5.0% TRF. As noted above, the TRF concentration isdetermined by with respect to the amount of ammonium sulfate present.

[0157] In one specific embodiment of the invention, E. coli strain472T23, which requires threonine for growth, may be converted to athreonine producer using P1-mediated transduction to introduce thethreonine operon of E. coli strain ATCC Deposit No. 21277, which may beobtained from the American Type Culture Collection, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA. This thr operon composed of afeedback resistant aspartate kinase-homoserine dehydrogenase gene(thrA), a homoserine kinase gene (thrB), and a threonine synthase gene(thrC). This strain may then be subjected to one, two, three, four, ormore cycles of mutagenesis, as described above, followed by screening toidentify cells which produce increased quantities of L-threonine orL-isoleucine.

[0158] To increase threonine production, the defective threoninedehydrogenase gene from E. coli strain CGSC6945 (relevant genotype:tdh-1::cat1212; obtained from the E. coli Genetic Stock Center, 355Osborne Memorial Laboratory, Department of Biology, Yale University, NewHaven, Conn. 06520-8104, USA) may be introduced into the cells by P1transduction. Again, the resulting threonine producer may be furtherimproved by mutagenesis followed by the identification of cells whichproduce increased amounts of L-threonine or L-isoleucine.

[0159] Plasmids carrying an antibiotic resistance marker gene, such askan (which encodes for kanomycin resistance), and a strong promoter,such as P_(L) or tac, optionally flanked by DNA upstream of thrA and afew hundred base pairs of the wild-type thrA gene (i.e., not the wholethrA gene), maybe constructed and used as a vehicle to deliver thedesired DNA fragment into the chromosome. The DNA fragment may beisolated by digestion with a suitable restriction enzyme and purified,and then introduced, through transformation or electroporation, into astrain to remove the control region of threonine operon and replace itby homologous recombination with the desired fragment (e.g., a fragmentcontaining an antibiotic resistance marker gene and a strong promoter atthe beginning the thrA gene). This fragment may then be transferred intothe cells of the strain by P1 transduction.

[0160] When increased production of L-threonine is desired, theisoleucine requirement of the strain of the one specific host, 472T23,maybe eliminated, for example, by introducing a wild-type allele of themarker through P1 transduction. Unwanted nutritional requirements ofother hosts may be removed in a similar manner or according to othermethods known and available to those skilled in the art.

[0161] Borrelidin- or CPCA-resistant strains of the invention maycontain one or more recombinant plasmids as desired. For example, theinventive microorganisms may contain recombinant plasmids that encodebiosynthetic enzymes of the threonine pathway. The inventive bacterialstrains may likewise contain recombinant plasmids encoding other enzymesinvolved in threonine biosynthesis, such as aspartate semialdehydedehydrogenase (asd), or enzymes which augment growth.

[0162] Additionally, the Borrelidin- or CPCA-resistant strains may bemodified as desired, for example, in order to increase threonineproduction, remove nutritional requirements, and the like, using any ofthe methods and techniques known and available to those skilled in theart. Illustrative examples of suitable methods for modifying Borrelidin-or CPCA-resistant E. coli mutants and variants include, but are notlimited to: mutagenesis by irradiation with ultraviolet light or X-rays,or by treatment with a chemical mutagen such as nitrosoguanidine(N-methyl-N′-nitro-N-nitrosoguanidine), methylmethanesulfonate, nitrogenmustard and the like; gene integration techniques, such as thosemediated by transforming linear DNA fragments and homologousrecombination; and

[0163] transduction mediated by bacteriophages such as P1. These methodsare well known in the art and are described, for example, in J. H.Miller, Experiments in Molecular Genetics, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A Short Course inBacterial Genetics, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.(1992); M. Singer and P. Berg, Genes & Genomes, UniversityScience Books, Mill Valley, Calif. (1991); J. Sambrook, E. F. Fritschand T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); P. B.Kaufman et al., Handbook of Molecular and Cellular Methods in Biologyand Medicine, CRC Press, Boca Raton, Fla. (1995); Methods in PlantMolecular Biology and Biotechnology, B. R. Glick and J. E. Thompson,eds., CRC Press, Boca Raton, Fla. (1993); and P. F. Smith-Keary,Molecular Genetics of Escherichia coli, The Guilford Press, New York,N.Y. (1989).

[0164] The present invention also includes the use of borrelidin- orCPCA-resistant bacterial strains in fermentation processes for theproduction of L-threonine (e.g., borrelidin- or CPCA-resistant mutantsof E. coli). Specific embodiments of the invention include mutantderivatives of E. coli strain ADM Kat13, which was deposited at theAgricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604, USA, on Jun. 28, 1996 andassigned accession number NRRL B-21593 and is described in Wang et al.,U.S. Pat. No. 5,939,307. Thus, strain ADM Kat13 may be subjected to one,two, three, four, or more cycles of mutagenesis, as described above,followed by screening to identify cells which produce increasedquantities of L-threonine or L-isoleucine.

[0165] Borrelidin or CPCA resistance maybe determined by any of theaccepted methods known to those skilled in the art. For example,borrelidin- or CPCA-resistant strains can be isolated by plating thecandidate strains on minimal medium containing about 139 μM borrelidinor CPCA, as described in G. Nass and J. Thomale, FEBS Lett. 39:182-186(1974). In addition, borrelidin or CPCA resistance in certain strains ismanifested as a change in one or more phenotypic characteristics of thecells. For example, borrelidin-resistant mutants of E. coli strain 6-8and its derivatives appear round, rather than as rods. In such cases,evidence of a change in a phenotypic characteristic may be sufficient toadequately identify borrelidin-resistant strains.

[0166] The borrelidin- or CPCA-resistant mutants useful in thisembodiment of the present invention are capable of producing threonine.The genes that encode the threonine biosynthetic enzymes may be presenton the chromosome or contained in plasmids or mixtures thereof. Multiplecopies of these genes may also be present. For example, the genes thatencode the threonine biosynthetic enzymes maybe resistant to attenuationcontrol and/or encode feedback-resistant enzymes.

[0167] Further, borrelidin- or CPCA-resistant mutants may also besubjected to one or more cycle of mutagenesis, followed by screening toidentify cells having desired characteristics, as described above. Thus,the invention also includes borrelidin- or CPCA-resistant mutants ofE.coli which are also resistant to TRF.

[0168] In one embodiment, the borrelidin- or CPCA-resistant mutants ofthe present invention are modified so as to include a non-nativepromoter upstream from and in operable link with one or more of thegenes that encode the threonine biosynthetic enzymes, regardless ofwhether these genes are on the chromosome and/or contained in plasmids.

[0169] D. Strains of the Invention

[0170] Examples of organisms, in addition to E. coli, which can be usedto prepare strains of the invention which produce increased quantitiesof amino acids include Brevibacterium flavum, Brevibacteriumlactofermentum, Brevibacterium divaricatum, Brevibacteriumsaccharolyticum, Corynebacterium glutamicum, Corynebacteriumacetoacidophilum, Corynebacterium lilium, Corynebacterium melassecola,Microbacterium ammoniaphilum, and Serratia marcesens.

[0171] In many instances, the inventive bacterial strains are strains ofE. coli. Further, as noted above, the invention includes bacterialstrains (e.g., E. coli strains) which exhibit resistance to themacrolide antibiotic borrelidin or cyclopentanecarboxylic acid. Specificexamples of bacterial strains of the invention includeE. coli strainsADM Kat69.9 (NRRL B-30316), ADM TH14.97 (NRRLB-30317), ADMTH 21.97(NRRLB-30318), and ADMTH25.79 (NRRL B-30319), each of which weredeposited at the Agricultural Research Service Culture Collection(NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, on Jul.27, 2000.

[0172] Strains of the invention also include strains which have thecharacteristics of the deposited strains assigned accession number NRRLB-30316, NRRL B-30317, NRRL B-30318, and NRRL B-30319. Particularcharacteristics these strains are set out below in Example 7.

[0173] Further included within the scope of the invention are bacterialstrains that do not require any recombinant plasmids containing one, twoor more genes that encode threonine biosynthetic enzymes for threonineproduction (i.e., strains capable of producing threonine without theneed for one or more of the threonine biosynthetic enzymes to be encodedby genes contained in a recombinant plasmid).

[0174] The inventive strains may, of course, optionally containrecombinant plasmids as desired. For example, while such plasmids aregenerally not required for threonine production, the inventive strainsmay nevertheless contain recombinant plasmids that encode for threoninebiosynthetic enzymes in order to increase threonine production. Theinventive strains may likewise contain recombinant plasmids encodingother enzymes involved in threonine biosynthesis, such as aspartatesemialdehyde dehydrogenase (asd).

[0175] Strains of the invention also include strains which are resistantto TRF and other agents resistance to which correlates with increasedthreonine production (e.g., cysteine, threonine and methionine analogs;isoleucine antagonists; and purine analogues).

[0176] In certain embodiments, the strains of the invention do notinclude one or more of the following strains of E. coli : KY10935, ADMTH1.2, BKIIM B-3996, H-8460,ADMKat13, tac3, 6-8, 6-8tac3, and6-8tac3ile+. In other embodiments, the strains of the invention do notinclude Serratia marcescens strain T2000.

[0177] In many instances, the novel bacterial strains also have no aminoacid nutritional requirements for fermentative production of threonine(i.e., the cells do not require amino acids supplements for growth andthreonine production).

[0178] Alternatively, bacterial strains of the invention may requiremethionine or isoleucine for growth.

[0179] III. Use of the Strains of the Invention to Produce Amino Acids

[0180] The present invention is also directed to the use of theabove-described bacterial strains in fermentation processes for theproduction of amino acids, amino acids of the aspartate family inparticular. L-threonine and L-isoleucine, for examples, may be obtainedby culturing the inventive bacterial strains in a synthetic or naturalmedium containing at least one carbon source, at least one nitrogensource and, as appropriate, inorganic salts, growth factors and thelike.

[0181] Illustrative examples of suitable carbon sources include, but arenot limited to: carbohydrates, such as dextrose, fructose, sucrose,starch hydrolysate, cellulose hydrolysate and molasses; organic acids,such as acetic acid, propionic acid, formic acid, malic acid, citricacid, and fumaric acid; and alcohols, such as glycerol and ethanol.

[0182] Illustrative examples of suitable nitrogen sources include, butare not limited to: ammonia, including ammonia gas and aqueous ammonia;ammonium salts of inorganic or organic acids, such as ammonium chloride,ammonium phosphate, ammonium sulfate and ammonium acetate; and othernitrogen-containing, including meat extract, peptone, corn steep liquor,casein hydrolysate, soybean cake hydrolysate and yeast extract. Culturemedia suitable for use with the present invention include the following:

[0183] 1. Minimal Medium E (described below in Example 1).

[0184] 2. Yeast extract 2 g/L, citric acid 2 g/L, (NH₄)₂SO₄25 g/L,KH₂PO₄ 7.46 g/L, CaCO₃ 20 g/L, dextrose 40 g/L, and MgSO₄.7H₂O2 g/L,supplemented with trace metals, pH 7.2.

[0185] 3. Yeast extract 5 g/L and tryptic soy broth 30 g/L.

[0186] Amino acids maybe commercially produced using strains of theinvention in, for example, batch type or fed-batch type fermentationprocesses. In batch type fermentations, all nutrients are added at thebeginning of the fermentation. In fed-batch or extended fed-batch typefermentations one or more nutrients are supplied (1) continuously to theculture, (2) right from the beginning of the fermentation or after theculture has reached a certain age, or (3) when the nutrient(s) which arefed are exhausted from the culture medium.

[0187] A variation of the extended batch of fed-batch type fermentationis the repeated fed-batch or fill-and-draw fermentation, where part ofthe contents of the fermentor is removed at a particular time (e.g.,when the fermentor is full) while feeding of a nutrient is continued. Inthis way a fermentation can be extended for a longer time as compared towhen such methods are not used.

[0188] Another type of fermentation, the continuous fermentation orchemostat culture, uses continuous feeding of a complete medium, whileculture fluid is continuously or semi-continuously withdrawn in such away that the volume of the broth in the fermentor remains approximatelyconstant. A continuous fermentation can in principle be maintained foran infinite period of time.

[0189] In a batch fermentation, the cultured organism grows until eitherone of the essential nutrients in the medium becomes exhausted orfermentation conditions become unfavorable (e.g., the pH decreases to avalue inhibitory for microbial growth). In fed-batch fermentationsmeasures are normally taken to maintain favorable growth conditions(e.g., by using pH control) and exhaustion of one or more essentialnutrients is prevented by feeding these nutrient(s) to the culture.Thus, the cultured microorganism will normally continue to grow at arate determined by the rate of nutrient feed.

[0190] In most instances, a single nutrient, very often the carbonsource, will become limiting for growth. The same principle appliesduring continuous fermentation, usually one nutrient in the medium feedis limiting and all of the other nutrients are in excess. After themicroorganisms have stopped growing, the limiting nutrient willgenerally be present in the culture fluid in an extremely lowconcentration.

[0191] While different types of nutrient limitation can be employed,carbon source limitation is used most often. Other examples are limitingnutrients include the nitrogen, sulfur, phosphorous, trace metal, andoxygen sources.

[0192] Vitamins and amino acid (in cases where the microorganism beingcultured is auxotrophic for the limiting amino acid) can also belimiting nutrients.

[0193] After cultivation, amino acids (e.g., L-threonine orL-isoleucine) that have accumulated in the culture broth can beseparated according to a variety of methods. For example, ion-exchangeresins according to purify L-threonine according to methods described inU.S. Pat. No. 5,342,766. This method involves first removing themicroorganisms from the culture broth by centrifugation and thenadjusting the pH of the broth to about 2 using hydrochloric acid. Theacidified solution is subsequently passed through a strongly acidiccation exchange resin and the adsorbent eluted using dilute aqueousammonia. The ammonia is removed by evaporation under vacuum, and theresulting solution is condensed. Addition of alcohol and subsequentcooling provides crystals of L-threonine. As similar method for thepurification of L-isoleucine from culture media is described in U.S.Pat. No. 5,474,918.

[0194] Other amino acids of the aspartate family can be produced bymethods similar to those described above. Isoleucine, for example, canbe prepared from the inventive bacterial strains containing, on thechromosome or on a plasmid, an amplified ilvA gene or tdc gene, both ofwhich encode threonine deaminase, the first enzyme involved in thebioconversion of threonine to isoleucine.

[0195] Amplification of this gene, for example, by use of a ilvA geneencoding a feedback-resistant enzyme, leads to increased biosynthesis ofisoleucine.

[0196] Similarly, methionine can be prepared by microorganisms such asE. coli that contain at least one met operon on the chromosome (i.e.,the metL gene (which encodes AK II-HD II), the metA gene (homoserinesuccinyltransferase), the metB gene (cystathionine γ-synthase), the metCgene (cystathionine β-lyase), and the metE and metH genes (homocysteinemethylase)). These genes, including feedback-resistant variants thereof,and, optionally, a non-native promoter can be introduced into thechromosome of the host microorganism according to general methodsdiscussed above and/or known to those skilled in the art. Lysine canlikewise be prepared by microorganisms that contain a gene encoding thelysine biosynthetic enzymes (e.g., a feedback-resistant lysinebiosynthetic enzyme encoded by lysC and/or dapA) and, optionally, anon-native promoter.

[0197] The present invention also includes the use of borrelidin- orCPCA-resistant bacterial strains in fermentation processes for theproduction of L-threonine (e.g., borrelidin- or CPCA-resistant mutantsof E. coli).

[0198] In specific embodiments of the present invention, L-threonine orL-isoleucine is obtained by culturing borrelidin- or CPCA-resistantmicroorganisms in a synthetic or natural medium containing at least onecarbon source, at least one nitrogen source and, as appropriate,inorganic salts, growth factors and the like, as described above. Aminoacids which accumulate in the culture media can be recovered by any ofthe methods known to those skilled in the art.

[0199] The following examples are illustrative only and are not intendedto limit the scope of the invention as defined by the appended claims.It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods of the presentinvention without departing from the spirit and scope of the invention.Thus, it is intended that the present invention cover the modificationsand variations of this invention provided they come within the scope ofthe appended claims and their equivalents.

[0200] All patents and publications referred to herein are expresslyincorporated by reference.

EXAMPLE 1 Preparation of E. coli Strain ADM Kat13

[0201] A. Transfer of the threonine operon of E. coli strain ATCCDeposit No. 21277 into the chromosome of E. coli strain 472T23.

[0202]E. coli strain ATCC Deposit No. 21277 (U.S. Pat. No. 3,580,810),available from the American Type Culture Collection, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA, is amino-β-hydroxyvaleric acid(AHV) resistant but requires proline, thiamine, isoleucine, andmethionine to grow in a minimal medium. ATCC Deposit No. 21277 isreported to accumulate 6.20 g/L of threonine in a fermentation process.The threonine operon of ATCC Deposit No.21277 is composed of anaspartate kinase 1-homoserine dehydrogenase I gene (thrA) that encodes afeedback-resistant enzyme, a homoserine kinase gene (thrB), and athreonine synthase gene (thrC).

[0203]E. coli strain 472T23, which is deposited in the USSR Collectionof Commercial Microorganisms at USSR Antibiotics Research Instituteunder Reg. No. BKIIM B-2307, is reported to require threonine andisoleucine and to grow in a minimal medium which contains glucose,ammonia, vitamin B1, and mineral salts. This strain cannot producethreonine because it carries a defective thrC gene, an essential genefor threonine biosynthesis. The strain 472T23 also carries a defectivethreonine deaminase gene, ilvA, which codes for the first enzyme inisoleucine biosynthesis.

[0204] Bacteriophage P1 lysate was prepared by growing phage on ATCCDeposit No.21277. Strain 472T23 was then infected with this P1 lysate,in which a small number of the phage particles carried the threonineoperon of ATCC Deposit No. 21277. Following infection, bacteriasynthesizing threonine were selected by spreading on minimal medium E(glucose 0.05 g/L; MgSO₄7H₂O 0.2 g/L; citric acid H₂O 2.0 g/L; K₂HPO₄10.0 g/L; NaHNH₄PO_(.4)4H₂O 3.5 g/L; agar 15.0 g/L) agar platessupplemented with 0.25 g/L isoleucine. Several threonine prototrophictransductants, which carried the threonine operon of ATCC Deposit No.21277, were now able to grow in a minimal plates supplemented only withisoleucine.

[0205] These transductants were screened by shake-flask fermentation forthreonine production as described below in Example 2. One of them, G9,producing threonine, was selected for further strain development.

[0206] B. Transfer of a defective threonine dehydrogenase (tdh⁻) geneinserted with a chloramphenicol acetyltransferase (cat) gene into thechromosome of E. coli strain G9.

[0207] Strain CGSC6945, carrying a defective threonine dehydrogenasegene (tdh⁻), was obtained from the E. coli Genetic Stock Center, 355Osborne Memorial Laboratory, Department of Biology, Yale University, NewHaven, Conn. 06520-8104, USA. The threonine dehydrogenase gene isdefective because inserted into it is the chloramphenicolacetyltransferase (cat) gene. To transfer this defective gene to G9, P1phage were grown on CSCG6945, and the lysate was used to infect G9.Several chloramphenicol-resistant transductants of G9 were selected andscreened for threonine production with shake-flask fermentation asdescribed below in Example 2. One of them, G909, with a higher threoninetiter than G9, was selected for further development.

[0208] C. Insertion of a non-native promoter into the chromosome of E.coli strain G909.

[0209] In order to deliver the tac promoter into the chromosome of G909,homologous recombination between a linear DNA fragment and thechromosome of an exonuclease V minus strain (recD) was employed.

[0210] The linear DNA fragment contained 1.5 kb of the sequence upstream(5′ end) of the threonine operon, a kanamycin resistant marker, the tacpromoter sequence, and about 480 bp of the thrA gene. This fragment,which provided 5′ end homology, a selection marker (kanamycinresistance), a strong and controllable promoter to the threonine operon(tac), and 3′ end homology, respectively, was generated as follows.

[0211] The threonine operon of the wild-type E. coli W3110 was clonedinto the restriction enzyme SphI site of plasmid pUC19 by using the DNAof the lambda clone 676 from Dr. Yuji Kohara, Department of MolecularBiology, School of Science, Nagoya University, Chikusa-ku, Nagoya,Japan. The DNAs of lambda clone 676 and pUC19 were then digested withSphI. The pUC19 fragment was subsequently dephosphorylated with shrimpalkaline phosphatase (SAP) and agarose-gel purified. The 6.9 kb fragmentof threonine operon from lambda clone was also purified. These twofragments were subsequently ligated by T4 DNA ligase to generate plasmidpAD103.

[0212] An upstream flanking region for homologous recombination andkanamycin resistance marker was then constructed. pAD103 was digestedwith restriction enzyme BstEII, XbaI and blunt-ended with klenowfragment treatment. The 1.5 kb fragment containing only the 5′ end(upstream) of the threonine operon (but not the thr operon itself or itscontrol region) was isolated and ligated to the fragment of kanamycinresistance gene from pUC4K (Pharmacia), which was digested withrestriction enzyme SalI and klenow fragment treated to fill-in the 3′overhangs to generate intermediate plasmid pAD106.pAD103 was alsodigested with restriction enzyme TaqI and blunt-ended with klenowfragment treatment. The fragment containing the native ribosome bindingsite and about 480 bp of the coding sequence of the thrA gene wasisolated and then ligated to a fragment of pKK233-3 (Pharmacia), whichhad been digested with restriction enzyme SmaI and dephosphorylated withSAP, to obtain plasmid pAD 115, which contained the DNA sequence of thetac promoter, the ribosome binding sites and a few hundred bases of thethrA gene.

[0213] pAD115 was subsequently digested with restriction enzyme BamHIand 0.75 kb of the DNA fragment which contained the desired DNAsequences was isolated. pAD106 was also digested with BamHI and thendephosphorylated with SAP. The two fragments were then ligated toprovide plasmid pAD123, which contained the DNA sequence upstream of thethreonine operon, a kanamycin resistance marker gene, the tac promoter,and about 480 bp of the beginning of the thrA gene.

[0214] pAD123 was then digested with SpeI, BgII and the fragmentcontaining the desired DNA sequences was isolated.

[0215] The exonuclease V minus strain (recD) was prepared by growing P1phage on E. coli strain KW251 (relevant genotype: argA81::Tn10,recD1014; obtained from Pharmacia), which contains a recD gene with aco-transducible transposon Tn10 insertion in argA. The lysate which wasprepared from the phage was then used to infect strain G9 and thetetracycline-resistant transductant G9T7 was isolated.

[0216] The DNA fragment from plasmid pAD123 was delivered to E. colistrain G9T7 by electroporation. A kanamycin-resistant strain of G9T7 wasisolated and a P1 phage lysate was made by growing phage on this strain.The P1 phage lysate was then used to transduce G909. One of thekanamycin-resistant transductants of G909, tac3, which showed a higherthreonine titer in the presence of IPTG in shake-flask study, wasisolated.

[0217] P1 phage lysate was subsequently prepared with strain tac3 andthen used to infect strain 6-8 (described below). Thekanamycin-resistant transductants were selected and one of them, strain6-8tac3, which produced an even higher titer than tac3 in a shake-flaskstudy, was isolated.

[0218] D. NTG mutagenesis and the isolation of borrelidin-resistantmutants from E. coli strains G909 and 6-8.

[0219] The cells of strain G909 were mutagenized byN-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment (50 mg/L, 30 min.at 36° C.) using conventional methods. The resulting cells were thenspread on minimal medium E agar plate containing 0.25 g/L ofL-isoleucine and 0.1% v/v of CPCA. After incubation for 3-5 days at 36°C., the large colonies that formed on the plate, which included strain6-8, were selected for testing for CPCA resistance and L-threonineproduction.

[0220] To test for CPCA resistance, each strain was cultivated in 20 mlof the seed medium SM (32.5 g/L glucose; 1 g/LMgSO₄.7H₂O; 24.36 g/LK₂HPO₄; 9.52 g/L KH₂PO₄; 5 g/L (NH₄)₂SO₄; 15 g/L yeast extract; pH 7.2)at 36° C. for 17 hr with shaking. The cells were harvested and washedwith minimal medium E. The cell suspension was then inoculated into asterilized tube containing 3 ml of minimal medium E and 0,0.1, 0.5, or 1mM CPCA. After 24 hr cultivation at 36° C. with shaking, growth wasdetermined by measuring the optical density at 660 nm. The results areshown below in Table 1 relative to growth in the absence of CPCA. TABLE1 CPCA (mM) G909 6-8 0 100.0 100.0 0.1 24.2 134.5 0.5 2.9 141.0 1 0.9184.5

[0221] E. Removal of isoleucine requirement and lactose repressor gene(lacI).

[0222] By introducing the non-native tac promoter and afeedback-resistant thrA gene, expression of the thr operon (thrA, thrB,thrC) is no longer controlled by the attenuation mechanism. As a result,starvation for isoleucine and/or the presence of an ilvA⁻ auxotrophicmarker is no longer required for threonine production.

[0223] Accordingly, the wild-type ilvA marker was introduced bytransduction into 6-8tac3 to fix the isoleucine requirement of thestrain (i.e., to eliminate the need for isoleucine-supplemented mediumfor cell growth). P1 phage lysate made from CGSC7334 (relevant genotype:lacI42::Tn10, lacZU118; obtained from the E. coli Genetic Stock Center,355 Osborne Memorial Laboratory, Department of Biology, Yale University,New Haven, Connecticut 06520-8104, USA) was used to infect 6-8tac3 andtransductants positive for isoleucine biosynthesis were selected. Thesetransductants produced approximately the same amount of L-threonine asstrain 6-8tac3 in a shake-flask study. One of these transductants,6-8tac3ile+ was selected for further development.

[0224] Since the threonine operon of 6-8tac3ile is under the control ofthe tac promoter, isopropyl-β-D-thiogalactoside (IPTG) was necessary toinduce the cells to fully express the thr operon.

[0225] Accordingly, to eliminate this unnecessary regulatory hindrance,a defective lac repressor (lacI) gene is introduced by infecting6-8tac3ile+with P1 phage made from CGSC7334. The resultant transductants(6-8tac3lacI-) were tested for resistance to tetracycline andtetracycline-resistant colonies were selected.

EXAMPLE 2 Shake-Flask Fermentation Study of Threonine Production

[0226] A comparison of threonine production among the various E. colistrains was determined by their performance in shake-flask fermentation.The strains being tested were grown on LB agar medium (10 g/L oftryptone, 5 g/L of extract, 15 g/L agar). After 1 to 2 days of growth,the cells were suspended in 5 ml of seed medium (dextrose 32.5 g/L;K₂HPO₄ 24.35 g/L; KH₂PO₄ 9.5 g/L; yeast extract 15 g/L; (NII₄)₂SO₄ 5g/L; MgSO₄.7H2O 1 g/L) at pH 7.2. The seed was grown for 24 hours with astirring speed of 250 rpm at 37° C. 15 ml of fermentation medium(dextrose 40 g/L; yeast extract 2 g/L; citric acid 2 g/L; (NH₄)₂SO₄ 25g/L; MgSO₄.7H₂O 2.8 g/L; CaCO₃ 20 g/L; trace metal solution 2 ml) at pH7.2 was then added to the seed and the fermentation process performed at37° C. with a stirring speed of 250 rpm. After cultivation, the amountof L-threonine that had accumulated in the culture broth was analyzed byHPLC (ISCO Model 2353 pump, Rainin Model RI-1 refractive index detector,and aminex Hp87-CA column).

[0227] The amount of L-threonine produced by each of the tested strainsis presented in Table 2 below. TABLE 2 Strain L-Threonine Produced (g/L)G909  4.95 6-8 11.45 tac3 12.9 (induced by IPTG) 10.6 (non-induced) 6-8tac3 ile+ 12.7 (induced by IPTG) 6-8 tac3 lacI− 13.9 ADM Kat13 14.0

EXAMPLE 3 Fermentation Study

[0228] The E. coli strains of the present invention and their precursorstrains were tested for L-threonine production by fermentation.

[0229] G909 was tested under the following conditions. 0.5 L of aqueousculture medium containing 30 g/L of tryptic soy broth and 5 g/L of yeastextract in a 2 L baffled shake flask was inoculated with 1.5 ml of G909and incubated on shaker at 35° C. and 200 rpm for 8.5 hours. 0.9 ml(0.03%) of the mature inoculum was added to a glass fermentor containing3.0 L of the seed fermentor medium (10 g/L d.s. of corn steep liquor,0.4 g/L of L-isoleucine, 2.5 g/L of KH₂PO₄, 2.0 g/L of MgSO₄.7H2O , 0.5g/L of(NH₄)₂SO₄, 0.192 g/L of anhydrous citric acid, 0.03 g/L ofFeSO₄.7H₂O, 0.021 g/L of MnSO4.H2O and 80 g/L of dextrose). Incubationwas conducted under the following conditions: a temperature of 39° C.for the first 18 hours, and then 37° C. for the duration; pH of 6.9(maintained by addition of NH₄OH); air flow of 3.5 LPM; agitation of 500rpm initially, which was then increased to maintain the D.O. at 20%; andback pressure of 1-2 psi. Completion of the seed fermentor stage wasdetermined by depletion of dextrose. 315 ml (15%) of the mature inoculumfrom the seed fermentor was added to a glass fermentor containing thesame medium (main fermentor medium) listed above with the followingexceptions: volume was 2.1 L and 0.34 g/L of L-isoleucine was added.Incubation was conducted for 48 hours under the following conditions:temperature of 37° C.; pH of 6.9 (maintained with NH₄OH); air flow of3.5 LPM until 20 hours then increased to 4.0 LPM; agitation of 500 rpminitially, which was then increased to maintain the D.O. at 20%; backpressure of 1-2 psi; and dextrose level of 10 g/L (maintained by feedingwith a 50% w/w dextrose solution). The fermentation was terminated after48 hours. G909 produced the following results: a final titer of 62.3 g/Lof threonine with a total productivity of 274 g and a yield of 23.2%.

[0230] tac3 was tested under the same conditions as described above forG909 with the following exception: 1 mg/L of IPTG was added at the startof the main fermentor stage. With addition of IPTG, tac3 produced afinal titer of 85.7 g/L of threonine with a total productivity of 355 gand a yield of 28.8%.

[0231] 6-8 was tested under the same conditions as G909 described above.6-8 produced the following results: a final titer of 74.1 g/L threoninewith a total productivity of 290 g and a yield of 28.3%.

[0232] 6-8tac3 was tested under the same conditions as tac3 describedabove, including the addition of IPTG. 6-8tac3 produced the followingresults: a final titer of 99.3 g/L threonine with a total productivityof 421 g and a yield of 35.1%.

[0233] 6-8tac3ile+ was tested under the same conditions as 6-8tac3 asdescribed above, with the following exception: no L-isoleucine wasrequired in either the seed fermentor stage or the main fermentor stage.Due to an agitation failure at 22.5 hours, only the titer at 22 hourswas recorded (62 g/L threonine).

[0234] ADM Kat13 was tested under the same conditions as 6-8tac3 asdescribed above with the following exception: no IPTG was added. Underthese conditions, ADM Kat13 produced a final titer of 102 g/L threoninewith a total productivity of 445 g and a yield of 33.1%.

[0235] The relevant genotypes of the constructed strains, supplementsrequired for fermentative production of threonine, and the titersrecorded are presented in Table 3. TABLE 3 Supplements Titer TiterRelevant for at 30 at 48 Strain Genotype Production Hours Hours Yield G9ilvA⁻, Ile ND ND ND G909 ilvA⁻, tdh::Cm Ile 53 62.3 23.2 tac3 ilvA⁻,tdh::Cm, Ile, IPTG 86 85.7 28.8 ptacthrABC 6-8 ilvA⁻, tdh::Cm, Ile 7074.1 28.3 Bor-R 6-8tac3 ilvA⁻, tdh::Cm, Ile, IPTG 75 99.3 35.1ptacthrABC, Bor-R 6-8tac3ile+ tdh::Cm, Bor-R, IPTG 62 NA NA ptacthrABC(at 22 hours) ADM Kat13 tdh::Cm, Bor-R, None   92.1 102   33.1ptacthrABC lacI⁻

EXAMPLE 4 Preparation of E. coli Strain ADM Kat69.9

[0236] A. Transfer of the Threonine Operon from an E. coli Strain, ADMKat26, into the Chromosome of E. coli Strain W3110

[0237] Strain ADM Kat26 has been constructed previously from E. coliATCC Deposit No. 21277 as shown in Table 4. The native threoninepromoter of this strain has been replaced by the tac promoter, at thesame time a kanamycin gene was introduced into the chromosome. A P1lysate was prepared by growing phage on ADM Kat26. Strain W3110 (ATCCDeposit No. 27325) was infected with this lysate, in which a smallnumber of the phage particles carried the threonine operon of ADM Kat26.Following infection, transfer of the threonine operon was selected foron rich media containing kanamycin. Several of these transductants werescreened in shake flask fermentation for threonine production, andinducibility of the threonine operon. One of the transductants, ADMKat60.6, was selected for further strain development.

[0238] B. Transfer of a defective threonine dehydrogenase (tdh⁻) geneinserted with chloramphenical acetyltransferase (cat) gene and anadditional copy of the threonine operon under the control of the tacpromoter into the chromosome of E. coli strain ADM Kat60.6

[0239] In order to introduce a second copy of the threonine operon intothe chromosome, a vector was constructed which knocked out the tdh geneby inserting a copy of the threonine operon. The first step in thisprocess was to construct a vector containing the appropriate genes. Thetdh::cat deletion from strain SP942 was cloned by digesting genomic DNAwith EcoRI, isolating the region of approximately 4.8 kb, and cloninginto the EcoRI site of plasmid puc 18 (FIG. 6). This plasmid was thendigested with Nael and the threonine operon with the kanamycin gene andtac promoter was cloned into the tdh gene (FIG. 7). This new constructwas linearized by digest with MluI and HindIII restriction enzymes. Thelinear piece containing the tdh with the second copy of the thr operonwas electroporated into a recD strain. Transformations were selected onrich media containing chloramphenical. A P1 lysate was made from one ofthese transformants, and was used to infect ADM Kat41, an ATCC DepositNo. 21277 derived threonine producer. A lysate was made from thisstrain, and this lysate was used to infect ADM Kat60.6. Thetransductants were selected on rich media containing chloramphenical.Shake flask studies were performed to screen for the best producer. Onestrain, ADM Kat68, was chosen for further manipulations.

[0240] C. Removal of the lactose repressor gene (lacI)

[0241] Since both threonine operons of ADM Kat68 are under the controlof the tac promoter, isopropyl-B-D-thiogalactoside (IPTG) was necessaryto induce the cells to fully express the thr operon. The use of IPTG toinduce expression of the thr operon is less preferred. To eliminate thisproblem, a defective lac repressor (lacI) gene was introduced byinfecting ADM Kat68 with P1 phage made from CAG 18439. All strainsinvolved in the construction of ADM Kat69.9 (NRRL B-30316) and theirgenotypes were shown in Table 4. The resultant transductants wereselected on rich media containing tetracycline, and then screened inshake flask for equal production of threonine with or without IPTG.TABLE 4 W3110 F⁻ mcrA mcrB IN(rrnD-rrnE)1 lambda⁻ ATCC 21277 pro, thi,iso, met SP942 F⁻, tdh−1::cat1212, IN(rrn-rrnE)1 CAG 18439 LacI/Tn10,lacZU118 ADM Kat26 kan-ptac-thrABC from tac3 transduced into Kat17 (ATCC21277 pro⁺, met⁺) ADM Kat41 Kat36.36(ATCC 2177 pro⁺, met⁺w.kan-ptac-thrABC from tac3; w.tdh-cm-ptac-thrABC, lacI::Tn10 from pIvirfrom Kat13) with homoserine resistance from Kat13 ADM Kat60.6 W3110 withkan-ptac-thrABC transduced from Kat26 ADM Kat68 Kat60.6 withtdh-cm-ptac-thrABC from Kat41 ADM Kat69.9 Kat68 with LacI::Tn10

EXAMPLE 5

[0242] Shake-Flask Fermentation Study of Threonine Production

[0243] A comparison of various E. coli strains was performed using theirproduction of threonine in the shake flask fermentation. The strainswere grown on LB agar media overnight, and then transferred to 20 mls ofshake flask media (dextrose 32.5 g/L; K₂HPO₄24.35 g/L; KH₂PO₄9.5 g/L;yeast extract 15 g/L; (NH4)₂SO₄ 5 g/L, MgSO₄.7H₂O 1 g/L) at pH 7.2. Theseed was grown for 24 hours with a stirring speed of 300 rpm at 37° C. 2ml of this cultured was transferred to the fermentation media (yeastextract 2 g/L; citric acid 2 g/L; (NH₄)₂SO₄ 25 g/L, KH₂PO₄.7.46 g/L;trace metal solution 2 ml/L; CaCO₃ 20 g/L; Dextrose 40 g/L; MgSO₄.7H₂O 2g/L) at pH 7.2. The fermentation was then run for 24 hours at 37° C. and300 rpm on a shaker. After cultivation, the amount of threonineaccumulated in the broth was analyzed by HPLC (as shown in Table 5).TABLE 5 Strain Threonine (g/L) Yield % ADM Kat60.6 4 14 ADM Kat68 7.5 19ADM Kat69.9 7.5 19

EXAMPLE 6 Mutagenesis and Selection for Mutants with ImprovedL-Threonine Production from Strain ADM Kat69.9

[0244] The cells of strain ADM Kat69.9 (NRRL B-30316) or its mutantswere harvested from mid-log phase cultures grown in LB, and thenmutagenized with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment(50 mg/L, 36° C, 25 minutes) in 3 ml of TM buffer (Tris.HCl 6.0 g/L,maleic acid 5.8 g/L, (NH₄)₂SO₄ 1.0 g/L, Ca(NO₃)₂ 5 mg/L, MgSO₄.7H₂O 0.1g/L, FeSO₄.7H₂O 0.25 mg/L, adjusted to pH 6.0 using KOH). After 25minutes of reaction, the NTG treated cells were pelleted bycentrifugation. The treated cells were washed twice in TM buffer andspread on minimal medium E (glucose 0.05 g/L, MgSO₄.7H₂O 0.2 g/L, citricacid H₂O 2.0 g/L, K₂HPO₄ 10.0 g/L, Na(HN₄)PO₄.4H₂O 3.5 g/L) agar platescontaining 4-8% of threonine or 0.2-0.5% of threonine raffinate (TRF)based on grams of ammonia sulfate per liter of medium, as determinedusing an ion sensitive probe which measures ammonium ions.

[0245] After incubation for 3-5 days at 36° C., colonies growing onthese plates were picked and tested for improved L-threonine productionin shaker flasks and fermentors. Mutants with improved threonineproduction were subjected to the next cycle of mutagenesis andselection. As shown in FIG. 8, strain ADM TH21.97 (NRRLB-30318) wasdeveloped from ADM Kat69.9 (NRRLB-30316) through the use of selectioncriterion designed to identify cells could grow faster, produce moreL-threonine in the formulated fermentation medium, and tolerate higherconcentrations of L-threonine and TRF as compared to their parentstrains.

EXAMPLE 7 Selection of Threonine Raffinate Mutants Strains

[0246] Both ADM TH14.97 (NRRLB-30317) and ADM TH25.79 (NRRLB-30319) aremutants which have been selected from E medium agar plates containing0.2-0.4% of TRF as described in Example 6. Strain ADM TH14.97 is a TRFmutant of ADM TH8.102 developed from ADM Kat69.9 (NRRL B-30316) asdescribed in FIG. 8. And strain ADM TH25.79 (NRRL B-30319) is a TRFmutant of ADM TH1.2 which was developed from ADM Kat13 (NRRLB-21593,U.S. Pat. No. 5,939,307). To study the effect of TRF on culture growth,selected TRF mutants and their parent strains were grown in mediacontaining TRF. About 0.1 ml culture prepared from each tested strainswas inoculated to a 250 ml baffled shaker flask containing 20 ml minimalmedium E and TRF at 0.1-0.4% based on grams of ammonia sulfate perlitter of medium. After shaking at 37° C. and 240 rpm for 24 hours,their growth O.D. was measured at 660 um. As shown in Table 6, ADMTH14.97 and ADM TH25.79 grew better with higher O.D. in minimal medium Econtaining TRF than their respective parent strains ADM TH8.102 and ADMTH1.2. TABLE 6 O.D. at 660 nm after growth in minimal medium E at 37° C.and 240 rpm for 24 hours. ADM ADM ADM ADM TRF TH8.102 TH14.97 TH1.2TH25.79 (%) (Parent) (TRF-R) (Parent) (TRF-R) 0.1 0.44 1.18 1.14 1.440.2 0.68 2.98 1.60 1.62 0.4 1.28 3.74 1.22 1.90

EXAMPLE 8 Dextrose Consumption, Growth, and L-Threonine Production InShaker Flask Fermentation

[0247] The L-threonine produced by E. coli strains was determined bytheir performance in the shaker flask fermentation. The strains beingtested were grown on LB agar medium (tryptophan 10 g/L, yeast extract 5g/L, NaCl 10 g/L, and agar 15 g/L). After 1 to 2 days of growth, cellswere inoculated to 20 ml seed medium A (K₂HPO₄ 24.36 g/L, KH₂PO₄ 9.5g/L, yeast extract 15 g/L, (NH₄)₂SO₄ 5 g/L, MgSO₄.7H₂O 1 g/L, dextrose32.5 g/L, pH 7.2) in a 250 ml baffled shaker flask. After growing at 37°C., 240 rpm shaking for 18 hours, 2 ml seed was inoculated into 20 ml offermentation medium A (dextrose 40 g/L, citric acid 2 g/L, lactose 1g/L, (NH₄)₂SO₄ 25 g/L, KH₂ PO₄.7.46 g/L, MgSO₄.7H₂O 2 g/L CaCO₃ 20 g/L,trace metal solution 2 ml/L, pH 7.2) in a 250 ml baffled shaker flask.After cultivation at 37° C., 240 rpm shaking for 24 hours, the amount ofL-threonine that had accumulated in the culture broth was analyzed byHPLC.

[0248] Under same incubation conditions indicated above, seed medium B(MgSO₄.7H₂O 2 g/L, (NH₄)₂SO₄ ₂₅ g/L, FeSO₄.7H₂O 0.03 g/L, MnSO₄H₂O 0.02g/L, KH₂ PO₄ 2.5 g/L, citric acid 0.2 g/L, corn steep liquor 20 g/L d.s.(dissolved solid), dextrose 40 g/L, CaCO₃ 40 g/L, pH 7.0) andfermentation medium B (MgSO₄.7H₂O 1.75 g/L, (NH₄)₂SO₄ 0.88 g/L, K₂HPO₄1.75 g/L, corn steep liquor 1.76 g/L d.s., dextrose 40 g/L, urea 20 g/L,CaCO₃ 17.5 g/L, pH 6.8) were also used in these studies to determine thethreonine production of selected mutants. Results of their L-threonineproduction and yield % in the shaker flask fermentation were shown inTable 7. TABLE 7 Seed/Fermentation L-Threonine Yield Strain Media (g/L)% ADM TH1.2 A/A  9.1 30.7 ADM TH25.79 A/A 13.0 31.4 ADM TH8.102 B/B 11.419.7 ADM TH14.97 B/B 11.6 25.1 ADM TH17.166 B/B 13.4 26.6 ADM TH21.97B/B 15.4 30.7

EXAMPLE 9 L-Threonine Production in Fermentor Fermentation

[0249] The L-threonine production of E. Coli strains was also determinedfrom their performance in fermentor fermentation. The strain beingtested was grown in a shaker flask medium containing 30 g/L of trypticsoy broth and 5 g/L of yeast extract. About 1.5 ml of culture wasinoculated into a 2 L baffled shake flask containing 0.5 ml shaker flaskmedium and incubated at 37° C. and 220 rpm for 8 hours. About 0.9 ml ofthe shaker flask culture was then transferred to a 5 L fermentorcontaining 3.0 L of the seed/main fermentor medium (corn steep liquor 10g/L d.s. (dissolved solids), KH2 PO₄ 2.5 g/L, MgSO₄.7H₂O 0.5 g/L,(NH₄)₂SO₄ 0.5 g/L, FeSO₄.7H₂O 0.03 g/L, MnSO₄H₂O 0.021 g/L, anhydrouscitric acid 0.192 g/L, dextrose 80 g/L). The cultivation of fermentorseed was conducted under following conditions: temperature at 39° C.,air flow at 3.5 LPM, agitation at 500 rpm initially, then increased tomaintain the D.O. at 20%, pH at 6.9 maintained by adding NH₄OH, and backpressure at 1-2 psi. After the completion of seed stage based on thedepletion of dextrose, 315 ml of seed culture was inoculated to another5 L fermentor containing 1.6 L of same seed/main fermentor medium asdescribed above. The fermentation was conducted for 48 hours under thefollowing conditions: temperature at 33° C., air flow at 3.5 LPM,agitation at 800 rpm initially, then increased to maintain the D.O. at20%, pH at 6.9 maintained by adding NH₄OH, and back pressure at 1-2 psi.The fermentation culture was fed with a 50% w/w dextrose solution tomaintain the dextrose level at 10 g/L in the fermentor. After 48 hours,samples were withdrawn to measure the amount of L-threonine producedusing HPLC (Table 8). TABLE 8 Relavent Titers L- Total L- Yield StrainPhenotype Threonine (g/L) Threonine (g) (%) ADM Kat69.9 Parent 5.1  12.9 2.9 ADM TH8.102 Thr-R 68.4 195.5 25.3 ADM TH14.97 Thr-R, 87.6 265.630.7 TRF-R ADM TH21.97 Thr-R, 96.2 292.2 35.5 TRF-R ADM TH1.2 Parent111.0 412.2 36.8 ADM TH25.79 TRF-R 117.3 442.8 37.4

We claim:
 1. A process for producing an Escherichia coli strainproducing between about 95 and about 150 g/L of L-threonine by about 48hours of growth in culture, said process comprising: (a) inserting intothe chromosome of an E. coli at least one threonine operon operablylinked to a non-native promoter to produce a parent strain; and (b)performing at least one cycle of mutagenesis on the parent strain,followed by screening the mutagenized cells to identify E. coli whichproduce between about 95 and about 150 g/L of L-threonine by about 48hours of growth in culture.
 2. The process of claim 1, wherein the E.coli strain produces between about 100 and about 140 g/L of L-threonineby about 48 hours of growth in culture.
 3. The process of claim 2,wherein the E. coli strain produces between about 110 and about 130 g/Lof L-threonine by about 48 hours of growth in culture.
 4. The process ofclaim 3, wherein the E. coli strain produces between about 110 and about120 g/L of L-threonine by about 48 hours of growth in culture.
 5. Theprocess of claim 1, wherein mutagenesis is performed using an agentselected from the group consisting of: (a) an alkylating agent; (b) anintercalating agent; and (c) ultraviolet light.
 6. The process of claim1, wherein two or three threonine operons are inserted into thechromosome of the E. coli.
 7. The process of claim 6, wherein theindividual threonine operons are operably linked to at least twodifferent non-native promoters.
 8. The process of claim 1, wherein thenon-native promoter is selected from the group consisting of the tacpromoter, the lac promoter, the trp promoter, the Ipp promoter, theP_(L) promoter and the P_(R) promoter.
 9. The process according to claim8, wherein the non-native promoter is the tac promoter.
 10. The processof claim 1, wherein the threonine operon contains a gene that encodes afeedback-resistant aspartate kinase-homoserine dehydrogenase.
 11. Theprocess according to claim 1, wherein the E. coli strain contains adefective threonine dehydrogenase gene on the chromosome.
 12. Theprocess of claim 1, wherein the threonine operon is obtained from thestrain deposited as ATCC Deposit No.
 21277. 13. The process of claim 1,wherein the mutagenized cells are screened to identify E. coli which areresistant to threonine raffinate.
 14. The process of claim 1, whereinthe mutagenized cells are screened to identify E. coli which areresistant to borrelidin.
 15. The process of claim 1, wherein themutagenized cells are screened to identify E. coli which are resistantto cyclopentanecarboxylic acid.
 16. The process of claim 1, wherein themutagenized cells are screened to identify E. coli which are resistantto threonine raffinate and borrelidin.
 17. The process of claim 1,wherein the mutagenized cells are screened to identify E. coli which areresistant to threonine raffinate and cyclopentanecarboxylic acid. 18.The process of claim 1, wherein the E. coli strain has thecharacteristics of the strain deposited as NRRL B-30318.
 19. The processof claim 1, wherein the E. coli strain has the characteristics of thestrain deposited as NRRL B-30319.
 20. An E. coli strain produced by theprocess of claim
 1. 21. An E. coli strain comprising at least onechromosomally integrated threonine operon operably linked to anon-native promoter, wherein said E. coli strain produces between about95 and about 150 g/L of L-threonine by about 48 hours of growth inculture, and wherein said E. coli strain is not strain KY10935, strainADM TH1.2, or strain ADM Kat13.
 22. The E. coli strain of claim 21 whichproduces between about 100 and about 140 g/L of L-threonine by about 48hours of growth in culture.
 23. The E. coli strain of claim 22 whichproduces between about 110 and about 130 g/L of L-threonine by about 48hours of growth in culture.
 24. The E. coli strain of claim 23 whichproduces between about 110 and about 120 g/L of L-threonine by about 48hours of growth in culture.
 25. The E. coli strain of claim 21comprising a threonine operon obtained from the strain deposited as ATCCDeposit No.
 21277. 26. The E. coli strain of claim 21 which is resistantto threonine raffinate.
 27. The E. coli strain of claim 21 which isresistant to borrelidin.
 28. The E. coli strain of claim 21 which isresistant to cyclopentanecarboxylic acid.
 29. The E. coli strain ofclaim 21 which is resistant to threonine raffinate and borrelidin. 30.The E. coli strain of claim 21 which is resistant to threonine raffinateand cyclopentanecarboxylic acid
 31. The E. coli strain of claim 21,where in said strain is selected from the group consisting of: (a) thestrain deposited as NRR B-30318; and (b) the strain deposited as NRRLB-30319.
 32. A process for producing L-threonine, which comprises thesteps of: (a) culturing an E. coli strain of claim 21 i n a culturemedium; and (b) recovering L-threonine from the culture medium.
 33. Theprocess of claim 32, wherein the E. coli strain produces between about100 and about 140 g/L of L-threonine by about 48 hours of growth inculture.
 34. The process of claim 33, wherein the E. coli strainproduces between about 110 and about 130 g/L of L-threonine by about 48hours of growth in culture.
 35. The process of claim 33, wherein the E.coli strain produces between about 110 and about 120 g/L of L-threonineby about 48 hours of growth in culture.
 36. The process of claim 32,wherein the non-native promoter is selected from the group consisting ofthe tac promoter, the lac promoter, the trp promoter, the lpp promoter,the P_(L) promoter and the P_(R) promoter.
 37. The process according toclaim 36, wherein the non-native promoter is the tac promoter.
 38. Theprocess of claim 32, wherein the threonine operon contains a gene thatencodes a feedback-resistant aspartate kinase-homoserine dehydrogenase.39. The process according to claim 32, wherein the E. coli straincontains a defective threonine dehydrogenase gene on the chromosome. 40.The process of claim 32, wherein the threonine operon is obtained fromthe strain deposited as ATCC Deposit No.
 21277. 41. The process of claim32, wherein the E. coli strain is resistant to threonine raffinate. 42.The process of claim 32, wherein the E. coli strain is resistant toborrelidin.
 43. The process of claim 32, wherein the E. coli strain isresistant to cyclopentanecarboxylic acid.
 44. The process of claim 32,wherein the E. coli strain is resistant to threonine raffinate andborrelidin.
 45. The process of claim 32, wherein the E. coli strain isresistant to threonine raffinate and cyclopentanecarboxylic acid. 46.The process of claim 32, wherein the E. coli strain has thecharacteristics of the E. coli strain deposited as NRRL B-30319.
 47. Theprocess of claim 32, wherein the E. coli strain has the characteristicsof a strain selected from the group consisting of: (a) the straindeposited as NRRL B-30318; and (b) the strain deposited as NRRL B-30319.
 48. The process of claim 32, wherein the E. coli strain is astrain selected from the group consisting of: (a) the strain depositedas NRRL B-30318; and (b) the strain deposited as NRRL B-30319.
 49. An E.coli strain which is resistant to threonine raffinate and producesbetween about 95 and about 150 g/L of L-threonine by about 48 hours ofgrowth in culture.
 50. The E. coli strain of claim 49 which producesbetween about 100 and about 140 g/L of L-threonine by about 48 hours ofgrowth in culture.
 51. The E. coli strain of claim 50 which producesbetween about 110 and about 130 g/L of L-threonine by about 48 hours ofgrowth in culture.
 52. The E. coli strain of claim 51 which producesbetween about 110 and about 120 g/L of L-threonine by about 48 hours ofgrowth in culture.
 53. The E. coli strain of claim 49, wherein thethreonine operon encodes a feedback-resistant aspartate kinase1-homoserine dehydrogenase I gene (thrA), a homoserine kinase (thrB)gene, and a threonine synthase gene (thrC).
 54. The E. coli strain ofclaim 49, wherein the threonine operon is obtained from the straindeposited as ATCC Deposit No.
 21277. 55. The E. coil strain of claim 49which contains a defective threonine dehydrogenase gene on thechromosome.
 56. The E. coli strain of claim 49 which is resistant toborrelidin or cyclopentanecarboxylic acid.
 57. The E. coil strain ofclaim 49 which has the characteristics of the strain deposited as NRRLB-30319.
 58. An E. coli strain selected from the group consisting of:(a) the strain deposited as NRRL B-30316; and (b) the strain depositedas NRRL B-30317.
 59. An E. coli strain having enhanced L-threonineproduction which is resistant to cyclopentanecarboxylic acid.